TECHNOLOGICAL FIELD

This invention relates to the field of detection of any target element or compound, particularly, pathogens, specifically viral pathogens. More specifically, the invention provides disposable low-cost devices, systems and methods for rapid, simple, selfsampling and self-performing detection of pathogenic agents in sample.

BACKGROUND ART

[1] Bruce, E. A. et al. Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step. bioRxiv, 2020.2003.2020.001008, doi: 10.1101/2020.03.20.001008 (2020).

[2] Zhang, Y. et al. Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv, 2020.2002.2026.20028373, doi: 10.1101/2020.02.26.20028373 (2020).

[3] Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28, e63-e63, doi:10.1093/nar/28.12.e63 (2000).

[4] Yu, L. et al. Rapid colorimetric detection of COVID-19 coronavirus using a reverse tran-scriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic plat-form: iLACO. medRxiv, 2020.2002.2020.20025874, doi: 10.1101/2020.02.20.20025874 (2020).

[5] Wang, X. et al. Rapid and sensitive detection of Zika virus by reverse transcription loop-mediated isothermal amplification. Journal of Virological Methods 238, 86-93, doi:https://doi.org/10.1016/j.jviromet.2016.10.010 (2016).

[6] Ben-Assa, N., Naddaf, R., Gefen, T., Capucha, T., Hajjo, H., Mandelbaum, N., Elbaum, L., Rogov, P., King, D.A., Kaplan, S. et al. Direct on-the-spot detection of SARS-CoV-2 in patients. Exp Biol Med (Maywood), 245, 1187-1193 (2020).

[7] Lafleur, K.L. et al., A rapid, instrument-free, sample-to-result nucleic acid amplification test. Lab Cip. 16, 3777-3787 (2016). BACKGROUND OF THE INVENTION

During a pandemic, surveillance is crucial for minimizing viral spread. In an attempt to contain the initial wave of the SARS-CoV-2 pandemic, countries went into lockdowns and actively tracked down new cases. These measures were drastic, yet worked effectively. The biggest flaw of the lockdown was its severe damage to the economy. After the lockdown was lifted and the economy reopened there was a rapid spread of the disease, also known as the ‘second wave’. Given the infected population size and its spread, it became no longer feasible to eradicate or contain the virus for long periods of time. Hence, the goal has shifted to devising a long-term strategy that will maintain public health while providing economic stability. The current solutions include creating moderate social distancing while testing for active cases in population-wide studies and direct testing of patients. However, although the intention is to actively test for new cases, in practice, this is extremely challenging. Current detection methods require professional teams, high-end equipment, and cumbersome logistics. Moreover, the time from sampling to results is at least several hours and up to 1-2 days. Most importantly, current detection methods imply exhaustion of isolation rooms in hospitals, waste of equipment and overwhelmed staff, as well as delays in diagnostics or interventions and delays in admission to high-risk non-COVID wards. All in all, managing suspected patients is a major challenge for hospitals, especially since most of the tested patients are negative. Detection of viral nucleic acids in patients is the gold standard detection method to date. It is currently performed at hospitals by professionals. As opposed to antibodies, detection of the viral RNA is a direct measure for the contagiousness of the patient. At this stage of the current COVID- 19 pandemic, it is clear that the availability and throughput of standard methods for viral nucleic acid detection is limited both by resources and accessibility to the community.

Standard detection methods for viral RNA in patients include RNA purification, reverse transcription and quantitative PCR (RT-qPCR). These processes are time consuming, require multiple biochemical reagents, lab-grade instruments and trained professionals [1]. Fortunately to date, alternative molecular biology methods can overcome these limitations. One of these methods is colorimetric Loop-Mediated Isothermal Amplification (LAMP) [2]. LAMP is performed at a single and constant temperature allows a one-step reverse transcription and its results can be visualized by color change. Due to the need for reverse transcription, this method is called reverse-transcribed (RT)- LAMP. These advantages reduce the need for sophisticated lab equipment [3-5].

The group of Naama Geva-Zatorsky and colleagues have recently demonstrated the use of loop-mediated isothermal amplification (LAMP) as a method for direct analysis of samples, including throat swabs, nasal swabs, and saliva [6].

Lafleur et al. [7], discloses diagnostic platform for performing a nucleic acid amplification test that requires no permanent instrument o manual sample processing, and involves sample collection and introduction, test activation and reading the results. The disclosed device prototype comprises a sample chamber for performing sample introduction and processing. The processed sample is delivered by an automated valve to a two-dimensional paper network (2DPN), where it is split into two physical channels. Isothermal amplification and lateral flow detection with gold nanoparticles is performed uncovered to facilitate flash photography. After amplification, a second valve operation releases the amplified lysate solutions that are transferred onto the detection zones. A simple device having in a single unit the sample preparation, the amplification reaction and the detection, as provided by the present invention, effectively reduces costs and simplifies the production and operation of such device.

There is therefore a need for simple portable, low cost LAMP diagnostic device allowing a rapid, one-step methods to provide results on-the-spot to detect viral nucleic acid materials from crude subject's samples, by non-professional untrained users in order to allow continuous surveillance of a community.

SUMMARY OF THE INVENTION

A first aspect of the present disclosure relates to sample testing module for use with a device for enabling detection and monitoring of at least one target element in at least one sample. In some examples of the disclosure, the sample testing module comprising a reaction pad and a support.

Moe specifically, the reaction pad of the sample testing module of the present disclosure, comprising a preparation zone and a plurality of reaction zones, each reaction zone being contiguous with the preparation zone via a respective branch element; wherein the preparation zone is configured for: (i) accommodating therein a first quantity of sample preparation agents, or a plurality of a first quantity of sample preparation agents, and at least a sample to be tested; (ii) enabling mixing of the first quantity of sample preparation agents (or a said plurality of the first quantity of sample preparation agents) with said sample to provide a prepared sample; (iii) accommodating a second quantity of a transporting liquid; and (iv) enabling contact of the prepared sample with said transporting liquid to provide a flowable prepared sample.

It should be noted that each of the reaction zone is configured for accommodating therein a respective third quantity of a respective reaction mixture adapted for said reaction, to produce at least one respective product, when reacted with a respective aliquot of the flowable prepared sample. Still further, the support of the disclosed module comprising a first heating system and a second heating system, the support being configured for supporting the reaction pad.

In some embodiments, the preparation zone, the plurality of reaction zones and the respective branch elements are integrally formed from a sheet of a first, a second and a third substrate materials, respectively, said first, second and third substrate materials configured at least for enabling delivery of a respective aliquot of said flowable prepared sample to each reaction zone under capillary action via the respective said branch element, said first, second and third substrate materials are either identical or different substrate materials.

It should be noted that in some embodiments of the present disclosure, each said reaction zone is configured for accommodating therein a respective third quantity of a respective reaction mixture adapted for the reaction, each said reaction zone being further configured for enabling reacting of each said respective aliquot of said flowable prepared sample with each said respective reaction mixture in the respective said reaction zone to produce at least one respective reaction product.

Still further, in some embodiments, the support of the sample testing module of the present disclosure, comprising a first heating system and a second heating system, the support being configured for supporting the reaction pad in nominally flat and overlying configuration with respect to the first heating system and the second heating system. In some embodiments, the first heating system is configured for enabling the preparation zone to be heated to at least one first temperature; wherein the second heating system is configured for enabling the amplification zones to be heated to a second temperature.

In yet some further embodiments, the first temperature is different from the second temperature.

In some embodiments, the target element detected and monitored by the disclosed testing module is at least one nucleic acid sequence of interest. In yet some further embodiments, the reaction performed in the reaction zones, is amplification reaction. In yet some further embodiments, the reaction zone/s as indicated herein are amplification zone/s.

In some embodiment, the applicator member will contain a volume smaller or equal to (but never larger than) the volume of the preparation zone. Thus, upon transfer of the sample, it will remain stationary on the preparation zone, until the transport of the transporting liquid is initiated.

In some embodiments, the sample preparation agents of the disclosed sample test module, comprise at least one of: (i) at least one proteolysis reagent; (ii) at least one surfactant; (iii) at least one chaotropic agent; and (iv) at least one solvent.

In some embodiments, the preparation zone of the sample testing module is configured for selectively allowing insertion of the sample into the preparation zone. In some particular and non-limiting embodiments, the preparation zone is configured for accommodating any appropriate sample volume. For example, a sample of between about 500pl or more, to about 5pl or less. In mor specific embodiments, the sample volume may be any one of 500pl or more, 450pl, 400pl, 350pl, 300pl, 250pl, 200pl, 150pl, lOOpl, 95pl, 90pl, 85pl, 80pl, 75pl, 70pl, 65pl, 60pl, 55pl, 50pl, 45pl, 40pl, 35pl, 30pl, 25pl. 20pl, 15 pl , lOpl, 5pl or less. In some specific embodiments, the preparation zone is configured for accommodating a sample volume of about 25 pl.

Still further, in certain embodiments, the reaction pad of the disclosed sample testing module, is made from at least one of a porous first, second and third substrate material having a pore size ranging between about 0.01 m to about 10pm. Specifically, any one of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.15, 0.16, 0.17, 0.18, 0.19, 0., 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10pm or more.

In some embodiments, the reaction pad of the disclosed sample testing module, has a length dimension, a width dimension, and a thickness dimension. In more specific embodiments, the thickness dimension of the reaction pad is significantly smaller than any one of the length dimension, and the width dimension.

In certain embodiments, the preparation zone of the disclosed sample testing module, has a first plan area, in yet some further embodiments, each amplification zone has a second plan area. In some specific embodiments, the summation of the second plan areas of the plurality of amplification zones is not greater than said first plan area. In some particular and non-limiting embodiments, the amplification zone is configured for accommodating any appropriate reaction volume that comprises the reaction reagents and enables the amplification reaction. For example, a reaction volume and/or reagents volume of between about 250 to about 1 .1. In mor specific embodiments, the reaction volume and/or reagents volume may be any one of 250pl or more, 200pl, 150pl, lOOpl, 95pl, 90pl, 85pl, 80pl, 75pl, 70pl, 65pl, 60pl, 55pl, 50pl, 45pl, 40pl, 35pl, 30pl, 25pl, 20pl, 15pl, lOpl, 9pl, 8pl. 7pl, 6pl. 5pl. 4pl, 3pl. 2pl, Ipl, or less. In some specific embodiments, the amplification zone is configured for accommodating a reaction volume and/or reagents volume of about 7 pl.

In some embodiments, the first plan area of the preparation zone of the disclosed testing module is about 400mm ² . In yet some further embodiments, each of the second plan area of each amplification zone is about 64mm ² .

In some embodiments, the preparation zone of the disclosed the sample testing module, is in the form of a first rectangle. In yet some further embodiments, each of the amplification zone is in the form of a respective second rectangle. In some specific embodiments, the preparation zone of the disclosed the sample testing module, is in the form of a first rectangle, and each of the amplification zone is in the form of a respective second rectangle.

In yet some further embodiments, the preparation zone of the disclosed sample testing module is in the form of a hexagon. In yet some further embodiments, each of the amplification zone of the disclosed sample testing module, is in the form of a respective cruciform. In more specific embodiments, the preparation zone of the disclosed sample testing module is in the form of a hexagon, and each of the amplification zone is in the form of a respective cruciform.

In yet some further embodiments, each of the branch element of the disclosed sample testing module is in the form of a respective strip connecting the respective amplification zone with the preparation zone.

In certain embodiments, the preparation zone of the disclosed sample testing module, comprises said plurality of sample preparation agents.

Still further, in some embodiments, the first heating system is configured for enabling the preparation zone of the disclosed sample testing module to be heated to at least one first temperature, in some specific embodiments, the first temperature is nominally 95°C +/- 5°C.

In yet some further embodiments, second heating system is configured for enabling the amplification zones of the disclosed sample testing module to be heated to at least one second temperature, in some specific embodiments, the second temperature is nominally 65°C+/-5°C.

In some particular embodiments, the first heating system of the support of the disclosed sample testing module, is configured for enabling the preparation zone to be heated to the first temperature for a predetermined first length of time. In some embodiments, the first length of time ranges between about 0.5 minute to about 5 minutes. Specifically, about 0.5 minutes or less, 0.6, 0.7, 0.8, 0.9, 0.1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5 minutes or more.

In yet some further particular embodiments, the second heating system of the support of the disclosed sample testing module, is configured for enabling the reaction zones to be heated to said second temperature for a predetermined second length of time.

In some embodiments, the second length of time ranges between about 20 minutes to about 50 minutes. Specifically, about 20 minutes or less, 25, 30, 35, 40, 45, 50 minutes or more.

In some embodiments, the first heating system of the support of the disclosed sample testing module, comprises a first heating element configured for being in at least one of radiative energy communication and conductive energy communication with the preparation zone.

In yet some further embodiments, the first heating element comprises at least one of: copper trace serpentine; conducting paste.

Still further, in some embodiments, the first heating element is electrically connected to a first power port comprised in the support. In some further embodiments, the first power port being selectively connectable to a power source.

In yet some further embodiments, the second heating system of the support of the disclosed sample testing module, comprises a plurality of second heating elements, each of these second heating element is configured for being in at least one of radiative energy communication and conductive energy communication with a respective said amplification zone of the disclosed module.

In some embodiments, at least one of: (a) the first heating element generates heat responsive to a first electric current being applied thereto; and (b) each of the second heating element generates heat responsive to a second electric current being applied thereto. Thus, in some embodiments, the first heating element generates heat responsive to a first electric current being applied thereto. In yet some further embodiments, each of the second heating element generates heat responsive to a second electric current being applied thereto. In some specific embodiments, the first heating element generates heat responsive to a first electric current being applied thereto, and each of the second heating element generates heat responsive to a second electric current being applied thereto.

In certain embodiments, each of the second heating element comprises at least one of: copper trace serpentine; and/or conducting paste.

In some embodiments, each of the second heating element is electrically connected to a second power port comprised in the support, the second power port being selectively connectable to a power source.

Still further, in some embodiments, the sample testing module of the present disclosure is further configured for enabling delivery of the second quantity of a transporting liquid to the preparation zone.

In some embodiments, the sample testing module of the preset disclosure further comprising: a reservoir pad formed from a sheet of fourth substrate material configured for receiving therein the second quantity of a transporting liquid; the support configured for supporting the reservoir pad in longitudinal spaced relationship with respect to the reaction pad by a gap, an electrically actuable microfluidic valve, having a normally closed configuration and an open configuration. More specifically, wherein: in the normally closed configuration any of the transporting liquid when present in the reservoir pad, is prevented by the gap from being transported to the preparation zone, and in the open configuration, any of the transporting liquid when present in the reservoir pad, is caused to be transported to the preparation zone via the gap, responsive to a third electrical current being supplied to the electrically actuable microfluidic valve. In yet some further embodiments, the sample testing module of the present disclosure is configured for selectively allowing insertion of the transporting liquid into the reservoir pad.

In some embodiments of the disclosed sample testing module, the reservoir pad is made from a porous fourth substrate material having a pore size ranging between about 0.01pm to about 10pm. More specifically, a pore size of between about 0.01pm or less, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10pm or more.

In yet some further embodiments, the respective gap is traversed by a connecting strip physically connecting the reservoir pad in longitudinal spaced relationship with respect to the reaction pad of the disclosed sample testing module.

In some embodiments, the connecting strip is configured to render the gap hydrophobic.

In some embodiments, the electrically actuable microfluidic valve of the disclosed sample testing module, comprises a positive electrode and a negative electrode spaced therefrom in a longitudinal direction. It should be understood that according to these embodiments, the positive electrode and the negative electrode being electrically connectable to a suitable electrical power supply, the positive electrode and the negative electrode being in contact with the transporting liquid. Still further, the gap is sealed with respect to at least a portion of the reservoir pad facing the gap, and with respect to at least a portion of the reaction pad facing the gap.

In some embodiments, the negative electrode is located closer to the gap than the positive electrode is located with respect to the gap.

In yet some further embodiments of the specific sample testing module discussed herein, the electrically actuable microfluidic valve comprises: a first hydrophobic electrode, a second hydrophobic electrode, and a hydrophobic insulating layer.

More specifically, in some embodiments, the first hydrophobic electrode being superposed on a first surface of the reaction pad spanning the gap and at least a portion of the reaction pad facing the gap, the second hydrophobic electrode being overlaid with respect to the hydrophobic insulating layer, the second hydrophobic electrode and the hydrophobic insulating layer being superposed on a second surface of the reaction pad, such that the hydrophobic insulating layer is sandwiched between the second surface of the reaction pad and the second hydrophobic electrode, and spanning the gap and at least a portion of the reaction pad facing the gap, the first hydrophobic electrode and the second hydrophobic electrode being electrically connectable to a suitable electrical power supply.

Thus, in operation the electrically actuable microfluidic valve is configured for generating a plasma discharge in said airspace in response to the third electrical current being provided to the electrically actuable microfluidic valve.

In some embodiments, the hydrophobic insulating layer comprises a plurality of through- holes.

In yet some further embodiments, the reservoir pad of the disclosed sample testing module, comprises said second quantity of said transporting liquid.

In yet some further embodiments of the sample testing module of the present disclosure, each of said plurality of amplification zones is configured for providing a respective amplification reaction under isothermal conditions. More specifically, the amplification reaction is according to some embodiments, a loop mediated isothermal amplification reaction (LAMP). Accordingly, in some embodiments, each respective reaction mixture is a LAMP reaction mixture, and each of the amplification zones comprises the respective LAMP reaction mixture.

In more specific embodiments, the first amplification zone of the plurality of the amplification zones comprises a first the LAMP reaction mixture. More specifically, the first amplification zone further comprises at least one set of primers specific for at least one nucleic acid sequence of the at least one nucleic acid sequence of interest.

In some embodiments of the sample testing module disclosed herein, the plurality of the amplification zones may comprise at least one of:

(a) a second amplification zone comprising a second said LAMP reaction mixture. The second amplification zone further comprises at least one set of primers specific for at least one nucleic acid sequence of at least one positive control; and

(b) a third amplification zone comprising a third said LAMP reaction mixture. The third amplification zone optionally further comprises at least one set of primers specific for at least one nucleic acid sequence of at least one negative control.

In some embodiments, the sample appliable for the disclosed sample testing module is at least one of a biological sample and an environmental sample.

In yet some further embodiments, the nucleic acid sequence of interest detected and monitored by the sample testing module of the present disclosure is a nucleic acid sequence of at least one pathogen.

In more specific embodiments, the pathogen is a viral pathogen.

In yet some further specific and nonlimiting embodiments, the viral pathogen detectable and/or monitored by the disclosed module is at least one corona virus (CoV), optionally, said CoV is Severe acute respiratory syndrome (SARS) CoV-2.

In yet some further embodiments of the disclosed sample testing module, wherein in operation of the sample testing module, the second quantity of a transporting liquid is externally delivered directly to the preparation zone.

Still further, in some embodiments, the sample testing module according to the present disclosure is configured as a single use disposable article, to be disposed after a single use. A further aspect of the present disclosure relates to a device for enabling detection of at least one pathogen in at least one sample, comprising: (a) a sample testing module as defined by the present disclosure; and (b) a control module configured for controlling operation of the device. It should be noted that the device being configured for enabling detecting a respective test parameter associated with said production of said at least one reaction product.

In some embodiments, the device of the present disclosure comprises a first upper device portion configured for being in overlying relationship with at least a first portion of the sample testing module. More specifically, the first upper device portion comprising at least one of: a sample receiving portal in registry with the preparation zone, at least in operation of the device, to enable insertion of the sample into the preparation zone; a transporting liquid portal in registry with the reservoir pad, at least in operation of the device, to enable insertion of the transporting liquid into the reservoir pad.

In some embodiments the device according to the present disclosure comprises an applicator member having a graspable handle and a sample receiving zone configured for enabling the sample to be delivered thereto from a user. The applicator member configured for being inserted through the sample receiving portal into liquid contact with the preparation zone to thereby enable the same to be transferred from the sample receiving zone to the preparation zone.

In yet some further embodiments, the disclosed device comprises a funnel arrangement circumscribing the transporting liquid portal.

In some further embodiments, the disclosed device comprises a detection module configured for enabling detecting of a detectable signal corresponding to a respective test parameter associated with said production of said at least one reaction product, in each of said plurality of reaction zones. In yet some further embodiments, the disclosed device comprises a second upper device portion configured for being in overlying relationship with at least a second portion of the sample testing module. More specifically, the second upper device portion comprising: an observation portal in registry with each said reaction zone, at least in operation of the device, to enable observation of a detectable signal corresponding to a respective test parameter associated with the respective reaction zone.

Still further, in some embodiments, the second upper device portion of the device disclosed herein comprises a plurality of illumination sources for illuminating the plurality of reaction zones at least in operation of the device.

In some embodiments, the control module of the device of the preset disclosure comprises a microcomputer chip configured for carrying out an automated detection test. More specifically, such detection test comprising the following steps:

(a) responsive to initiation of the test by the user, allowing at least the sample to be tested and the first quantity of at least one sample preparation agent to interact with one another for a first predetermined time period;

Next in step (b), following step (a), causing the first heating system to heat the preparation zone to the first temperature for a first time duration;

Next in step (c), following step (b), operating the electrically actuable microfluidic valve to cause the transporting liquid to be transported to the preparation zone via the gap and allowing the respective aliquot of each of the prepared sample to be transported to the respective reaction zone under capillary action via the respective branch element;

Next in step (d), following step (c), causing the second heating system to heat each of the reaction zone to the second temperature for a second time duration;

In step (e), following step (d), enabling detection of a detectable signal corresponding to a respective test parameter associated with the production of the at least one amplification product in each of the reaction zone.

In some embodiments, the device of the present disclosure comprises a power input port configured for enabling an electrical power connector to be connected thereto to enable electrical power from an external power source to be connected thereto, to thereby enable electrical power to be provided at least to the control module, the first heating system and the second heating system.

Still further, in some embodiments, the control module of the device of the present disclosure is integrated into the support of the sample testing module.

Still further, in some embodiments, the control module of the device disclosed herein is separate from the support of the sample testing module.

In yet some further embodiments, the device according to the present disclosure comprises a testing chamber configured for enabling the sample testing module to be selectively inserted into the device such as to be operatively coupled with the control module and such as to enable the first heating system and the second heating system to be selectively provided with electrical power, and for enabling the sample testing module to be selectively removed from the device after the test is completed. More specifically, the device is configured for ensuring that the testing chamber avoids contamination from the sample.

In some embodiments, the target element detected and monitored by the disclosed device is at least one nucleic acid sequence of interest. In yet some further embodiments, the reaction performed in the reaction zones, is amplification reaction. In yet some further embodiments, the reaction zone/s as indicated herein are amplification zone/s.

A further aspect of the present disclosure provides a system for enabling detection of at least one target element in one sample, for each one of a plurality of said samples, comprising at least a corresponding plurality of said devices as defined in the present disclosure, and an electrical supply operatively connected to each said device.

In a yet a further aspect, the disclosure provides a method for the detection and monitoring of at least one target element in at least one sample. In more specific embodiments, the method of the invention comprises the following steps.

The first step (i), involves applying at least one sample in a preparation zone of a device as defined by the invention. The application of the sample in the preparation zone, initiates the automated detection test. However, it should be appreciated that in some alternative or additional embodiments, a more direct initiation by the user may be required, for example in cases that a sample is not placed in the device. Thus, in some alternative or additional embodiments, the initiation step may involve any one of the following steps, placing the applicator member in the sample receiving zone of the device of the invention (that is configured for enabling the sample to be delivered thereto), pressing a button, and/or turning a switch on to initiate the reaction. In some optional embodiment, this initial step may further comprise providing a second quantity of a transporting liquid to the reservoir pad of the device.

The second step (ii), allows for an automated detection test, controlled by the control module. In more specific embodiments, the control module controls the following steps or actions:

In the reaction start step (a), responsive to initiation of the test by the user, allowing at least the sample to be tested and the first quantity of at least one sample preparation agent to interact with one another for a first predetermined time period. It should be understood that the initiation of the test by the user may involve any direct or indirect signal/s or action/s, for example, any one of the following steps, adding the sample, placing the applicator member in the sample receiving zone of the device of the invention (that is configured for enabling the sample to be delivered thereto), adding a transporting liquid to the reservoir, pressing a button, and/or turning a switch on to initiate the reaction.

In the next step (b), following step (a), causing the first heating system to heat the preparation zone to the first temperature for a first time-duration.

In the next step (c), following step (b), operating the electrically actuable microfluidic valve to cause the transporting liquid to be transported to the preparation zone via the gap and allowing the respective aliquot of each prepared sample to be transported to each respective reaction zone, under capillary action via the respective branch element.

In the next step (d), following step (c), causing the second heating system to heat each of the reaction zones of the plurality of amplification zones, to the second temperature for a second time duration to allow an amplification reaction.

In the next step (e), following step (d), enabling detection of a detectable signal corresponding to a respective test parameter associated with the production of at least one reaction product by the reaction in each reaction zone. The third step of the method of the invention (iii), involves determining the appearance of at least one test parameter associated with the production of a reaction product in each the amplification zones by detecting a detectable signal.

In some embodiments, the target element detected and monitored by the disclosed method is at least one nucleic acid sequence of interest. In yet some further embodiments, the reaction performed in the reaction zones, is amplification reaction. In yet some further embodiments, the reaction zones are amplification zones.

As indicated above, the first reaction zone, specifically, amplification zone comprises at least one set of primers specific for at least one nucleic acid sequence of the at least one nucleic acid sequence of interest. Therefore, the detection of a detectable signal in the first amplification zone of the plurality of amplification zones, indicates the presence of the nucleic acid sequence of interest in the sample.

In some embodiments, the second step of the method of the invention, specifically, the automated detection test that is controlled by the control module, may further comprise an additional preliminary step of sample preparation. According to such step, prior to step (a), the preparation zone, containing the sample, is preheated to said first temperature for a third time duration. This initial and optional step facilitates sample preparation, by leading to lysis of any proteins associated to the nucleic acid molecules in the sample. Such proteins may be part of biological membranes in case of a biological sample, for example, cellular membranes or viral envelops. This pre-heating step in some embodiments, may involved heating the preparation zone to a temperature ranging between about 50 to 80°C, specifically, 65°C+/-5°C, for thermal lysis.

As indicate above, the diagnostic method of the invention is based on amplification reaction to amplify the nucleic acid molecules in the sample. In some embodiments, the amplification reaction performed by the methods of the invention is an amplification reaction under isothermal conditions, optionally, a loop mediated isothermal amplification reaction (LAMP). It should be noted that in some embodiments, all amplification zones in the device used by the methods of the invention comprise an amplification reaction mixture, specifically, LAMP amplification reaction mixture. In yet some further embodiments, at least one amplification zone of the plurality of amplification zones further comprises reagents specific for detection of the nucleic acid sequence of interest. By way of example, an amplification zone that comprise reagents specific for detecting the nucleic acid sequence of interest, may be referred to herein as a "first" amplification zone. In yet some further embodiments, the first amplification zone of the plurality of amplification zones comprises a LAMP reaction mixture, and further reagents for detection of the nucleic acid sequence of interest. Thus, in some specific embodiments, the first amplification zone further comprises at least one set of primers specific for at least one nucleic acid sequence of the at least one nucleic acid sequence of interest.

In yet some further embodiments, the plurality of the amplification zones comprise in addition to the first amplification zone discussed above, also at least one amplification zone that comprise in addition to LAMP reaction mixture, also amplification reagents that are specific for at least one control nucleic acid sample. Such control reaction may be a positive or negative control, or both. By a way of example, the amplification zone that comprise these controls, may be referred to herein as a second and third amplification zones. Thus, in some embodiments, the device used by the methods of the invention may comprise at least one of:

(a), a second amplification zone that is designed to serve as a positive control, comprising a second LAMP reaction mixture. Such second amplification zone may therefore comprise in addition to LAMP reaction mixture, also at least one set of primers specific for at least one nucleic acid sequence of at least one positive control nucleic acid sequence; and

(b), a third amplification zone that is designed to serve as a negative control, comprising a third LAMP reaction mixture. Such third amplification zone may optionally comprise in addition to the third LAMP reaction mixture, also at least one set of primers specific for at least one nucleic acid sequence of at least one negative control nucleic acid sequence. In yet another option, a third amplification zone designed to serve as a negative control may comprise only a third LAMP reaction mixture with no primers. In yet some further embodiments, the sample examined by the methods of the invention is at least one of a biological sample and an environmental sample.

In yet some further embodiments, the nucleic acid sequence of interest may be a nucleic acid sequence of at least one pathogen. Thus, according to some embodiments, the method of the invention may be useful for the detection and monitoring of at least one pathogen in at least one sample.

In some embodiments, the pathogen may be at least one viral pathogen.

In yet some more specific embodiments, the viral pathogen is at least one CoV. In some particular embodiments, such CoV may be Severe acute respiratory syndrome (SARS) CoV-2. Thus, in accordance with some embodiments, the invention provides methods for the detection and monitoring of SARS CoV-2 in at least one sample.

These and other aspects of the present disclosure will become apparent by the hand of the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 is an isometric view of a device according to a first example of the presently disclosed subject matter; Fig. 1(a) is a side view of the applicator of the example of Figure. 1.

Fig. 2 is an isometric view of a reaction pad for use in the example of Fig. 1 according to an example of the presently disclosed subject matter. .

Fig. 3 is a top view of a sample testing module for use with the example of Fig. 1 according to an example of the presently disclosed subject matter.

Fig. 4 is a top view of a support of the example of the sample testing module of Fig. 3.

Fig. 5 is a top view of the example of Fig. 1. Fig. 6 is a cross-sectional view of an example of a valve corresponding to the device example of Fig. 3 taken along A-A.

Fig. 7 is a cross-sectional view of another example of a valve alternative to the example of Fig. 6.

Fig. 8 is an isometric view of a device according to a alternative variation of the example of Fig. 1.

Fig. 9 is a top view of the example of Fig. 8.

DETAILED DESCRIPTION

Referring to Fig. 1, a device 10 for enabling detection and monitoring of at least one nucleic acid sequence of interest in at least one sample according to a first example of the presently disclosed subject matter, comprises a sample testing module 100, a control module 900, and an upper cover 950. In particular the device 10 is configured for enabling detecting a respective test parameter associated with said production of said at least one amplification product. Optionally, the device can include a lower cover 990.

In this example the device 10 is configured as a one-use disposable device, to be discarded or otherwise disposed of after one use.

It is to be noted that at least in some examples, in use of the device 10 the sample can be preheated to a desired temperature for a desired time period.

Referring also to Figs. 2, 3 and 4, the sample testing module 100, for use with device 10 for enabling detection and monitoring of at least one nucleic acid sequence of interest in at least one sample according to a first example of the presently disclosed subject matter, comprises a reaction pad 200 and a support 300.

The reaction pad 200 comprises a preparation zone 210 and a plurality of amplification zones 230, each amplification zone 230 being contiguous with the preparation zone 210 via at least one respective branch element 220.

The preparation zone 210 is configured for each of the following:

(i) accommodating therein a plurality of a first quantity of sample preparation agents and at least a sample to be tested; (ii) enabling mixing of said plurality of a first quantity of sample preparation agents with said sample to provide a prepared sample;

(iii) accommodating a second quantity of a transporting liquid; and

(iv) enabling contact of the prepared sample with said transporting liquid to provide a flowable prepared sample;

Each amplification zone 230 is configured for accommodating therein a respective third quantity of a respective reaction mixture adapted for amplification reaction. Furthermore, each amplification zone 230 is further configured for enabling reacting of each respective aliquot of said flowable prepared sample with each said respective reaction mixture in the respective amplification zone 230 to produce at least one respective amplification product.

While in at least this example, the reaction pad 200 comprises three said amplification zones 230, in alternative variations of this example, the reaction pad 200 can instead comprise less than three said amplification zones 230, for example two said amplification zones 230, or more than three said amplification zones 230. In at least this example each amplification zone 230 is contiguous with the preparation zone 210 via one respective branch element 220, and thus in this example the reaction pad 200 comprises three branch elements 220. In some embodiment, the reaction pad of the disclosed device may comprise about to 50 or more amplification reaction ones, specifically, 1, 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amplification zones.

While in at least this example, the branch elements 220 are connected to the preparation zone 210 via a common branch element 235, in alternative variations of this example each amplification zone 230 is connected to the preparation zone 210 directly via its respective branch element without such a common branch element. Thus, the common branch element 235 projects from a side 211 (Fig. 2) of the preparation zone 210; one branch element 230 is longitudinally aligned with the common branch element 235, and the two other branch elements 230 branch off in opposed directions, transversely with respect to the common branch element 235. In at least this example, the reaction pad 200 is integrally formed from a sheet of substrate material, and thus the preparation zone 210, the amplification zones 230, the branch elements 220 (including the common branch 235) are integrally connected to one another. The substrate material is configured at least for enabling delivery of a respective aliquot of a flowable prepared sample to each amplification zone under capillary action via the respective said branch element, as will become clearer herein. For example, the reaction pad 200 is made from a porous substrate material having a pore size ranging between about 0.01pm to about 10pm, specifically, a pore size of between about 0.01pm or less, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10pm or more, for example about 0.45pm or about 0.8pm. In at least some examples, the reaction pad 200 is made from, i.e., the substrate material comprises, a 8-micron Polyethersulfone (PES) filter (hydrophilic and constructed from Polyethersulfone polymer membrane). In yet other examples, the reaction pad 200 is made from, i.e., the substrate material comprises, at least one of: nitrocellulose, vinylidene fluoride (PVDF), or glass fibers.

However, in alternative variations of this example, the preparation zone 210, the plurality of amplification zones 230 and the respective branch elements 220 (including the common branch 235) are integrally formed from a sheet of a first, a second and a third substrate materials, respectively, wherein each one of the first, second and third substrate materials is configured at least for enabling delivery of a respective aliquot of a flowable prepared sample to each respective amplification zone under capillary action via the respective said branch element, the first, second and third substrate materials are either identical or different substrate materials, as will become clearer herein. For example, the reaction pad 200 is made from at least one of a porous first, second and third substrate material having a pore size ranging between about 0.01pm to about 10pm, for example about 0.45pm or about 0.8pm In at least some examples, the reaction pad 200 is made from, i.e., one or more of the first, second and third substrate material comprises, a 8-micron PES filter. In yet other examples, the reaction pad 200 is made from, i.e., one or more of the first, second and third substrate material comprises, at least one of: nitrocellulose, PVDF, or glass fibers. Referring in particular to Fig. 2, the reaction pad 200 has a length dimension L (along the longitudinal direction with respect to the device 10 or the sample testing module 100), a width dimension W, and a thickness dimension T, the thickness dimension T being significantly smaller than any one of the length dimension L and the width dimension W. For example, the length dimension L can be in the range 100mm to 200mm, for example about 120mm; for example the width dimension W can be in the range 50mm to 100mm, for example about 60mm; for example the thickness dimension T can be less than 0.1mm in the range 0.01mm to 0.10mm, for example.

In at least this example, the preparation zone 210 has a first plan area Al, each amplification zone 230 has a second plan area A2, and the respective branch elements 220 (including the common branch 235) have a collective plan area of A3.

For example, first plan area Al can be in the range between about 100mm ² and about 900mm ² , for example 400mm ² . For example each second plan area A2 can be in the range between about 10mm ² and about 100mm ² , for example about 64mm ² .

In this example, the summation of the second plan areas A2 is not greater than said first plan area Al. In alternative variations of this example, the summation of the second plan areas A2 is about the same as the first plan area Al. In yet other alternative variations of this example, the summation of the second plan areas A2 greater than the first plan area Al .

In this example, the preparation zone 210 is in the form of a rectangle, and each amplification zone 230 is also in the form of a respective rectangle; furthermore, each branch element 220 (including the common branch 235) is in the form of a respective rectangular strip essentially connecting the respective amplification zone 230 with the preparation zone 210. However, in alternative variations of this example, the preparation zone 210, the amplification zones 230, and the branch elements 220 (including the common branch 235) can have any suitable size and/or shape. For example, and referring to Figs. 8 and 9, in an alternative variation of the example of Figs. 1 to 4, the respective preparation zone 210' is hexagonal, the respective amplification zones 230' are cruciform shaped, and the respective common branch 235' is trapezoidal, being integrally formed with the respective preparation zone 210'.

As will become clearer herein, and referring again to Figs. 1 to 4, the preparation zone 210 is configured for selectively allowing insertion of the sample into the preparation zone 210. In use, the preparation zone 210 comprises the aforesaid plurality of sample preparation agents. For example, the reaction pad 200 comes factory prepared with the plurality of sample preparation agents. Alternatively, the plurality of sample preparation agents can be delivered to the reaction pad 200 at any suitable time prior to conducting the sample testing with device 10.

In at least some examples, the plurality of sample preparation agents is embedded within the substrate material of the reaction pad 200.

In at least some examples, the sample preparation agents comprise at least one of: (i) at least one proteolysis reagent; (ii) at least one surfactant; (iii) at least one chaotropic agent; and (iv) at least one solvent. For example, the sample preparation agents can comprise at least one chaotropic agent (e.g., guanidine hydrochloride, that may be added in the preparation step and/or the amplification reaction step). In yet some further embodiments, the sample preparation agent may comprise at least one protease, specifically, at least one serine protease as discussed herein after.

Referring in particular to Fig. 3 and Fig. 4, the support 300 comprises a first heating system 420 and a second heating system 470.

The support 300 is configured for supporting the reaction pad 200 in a nominally flat and overlying configuration with respect to the first heating system 420 and the second heating system 470.

In at least this example, the support 300 is in the form of a rectangular block, having an upper face 302 and a lower face 304, spaced by the thickness dimension TB of the support 300.

In at least this example, the support 300 includes a first recess 320 in the upper face 302. The first recess 320 is sized and shaped to receive the reaction pad 200, and thus comprises a first recess portion 322 sized and shaped to receive the preparation zone 210, a plurality (in this example, three) of second recess portions 324 each sized, shaped and in spatial relationship with respect to the first recess portion 322 to receive the respective amplification zone 230, and a third recess portion 326 sized, shaped and in spatial relationship with respect to the first recess portion 322 and to the second recess portions 324 to receive the branch elements 220 (including the common branch 235).

The first heating system 420 is configured for enabling the preparation zone 210 to be heated to at least a first temperature Tl. In at least some embodiments the first temperature Tl is a temperature suitable for denaturation and inactivation of at least one of said sample preparation agent/s. For example, the first temperature Tl is nominally 95°C ±5°C. Furthermore, the first heating system 420 is configured for enabling the preparation zone 210 to be heated to said first temperature Tl for a predetermined first length of time. In at least this example, such a first length of time can be sufficient for denaturation and inactivation of at least one of said sample preparation agent/s. For example, such a first length of time can be within the ranges between about 0.5 minute to about 5 minutes.

Optionally, the first heating system 420 can be further configured for enabling the preparation zone 210 to be heated to a number of different temperatures, sequentially, wherein one such temperature is the first temperature Tl. For example, while the first temperature Tl can correspond to an inactivation temperature, for example about 95 °C, specifically, any one of 100°C or more , 99°C, 98°C, 97°C, 96°C, 95°C, 94°C, 93°C, 92°C, 91°C, 90°C or less, specifically, 95 °C, the first heating system 420 can be configured for enabling the preparation zone 210 to be heated first to a lower, lysis temperature, for example about 60 °C, specifically, any one of 65°C or more , 64°C, 63°C, 62°C, 61°C, 60°C, 59°C, 58°C, 57°C, 56°C, 55°C or less, 60 °C, in such examples, the first heating system 420 is configured for enabling the preparation zone 210 to be heated to each temperature of the aforesaid different temperatures sequentially, maintaining each such temperature for a corresponding predetermined length of time. For example, the first heating system 420 can heat the preparation zone 210 for a corresponding length of time to enable lysis, and then heat the preparation zone 210 at a different temperature, for example the first temperature Tl for a length of time can be sufficient for denaturation and inactivation of at least one of said sample preparation agent/s. For example, such lengths of time can be in the range between about 0.5 minute to about 5 minutes. The first heating system 420 comprises a first heating element 425 configured for being in at least one of radiative energy communication and conductive energy communication with the preparation zone 210. In other words, in operation, the first heating element 425 heats the preparation zone 210 via heat conduction and/or heat radiation between the first heating element 425 and the preparation zone 210. The first heating element 425 is accommodated in the first recess portion 322 and is electrically connected to a first power port 392 comprised in the support 300 (Fig. 1). The first power port 392 is selectively connectable to a power source (not shown). While in at least this example, the first heating element 425 comprises a copper trace, in alternative variations of this example the first heating element can comprise at least one of: conductive trace serpentine; conducting paste. For example, the copper trace serpentine has a width of about 0.5mm, and adjacent traces are separated by a non-conducting gap of about 0.5mm.

Thus, in operation of the first heating system 420, the first heating element 425 generates heat, and applies the hat to the preparation zone 210, responsive to a first electric current being applied thereto, provided thereto by the power source via first port 392.

The second heating system 470 is configured for enabling each one of the amplification zones 230 to be heated to a second temperature T2. In at least this example the first temperature T1 is different from the second temperature T2. In at least some embodiments the second temperature T2 is a temperature suitable for allowing an amplification reaction. For example, the second temperature T2 is nominally 65°C ±5°C.

Furthermore, the second heating system 470 is configured for enabling each of the amplification zones 230 to be heated to the second temperature T2 for a predetermined second length of time. For example, such a second length of time can be in the range of between about 20 minutes to about 50 minutes, for example 35 minutes.

The second heating system 470 comprises a plurality of second heating elements 475 (in this example, three second heating elements 475), each second heating element corresponding to a respective amplification zone 230. Each second heating element 475 is configured for being in at least one of radiative energy communication and conductive energy communication with the respective amplification zone 230. In other words, in operation, each second heating element 475 heats the respective amplification zone 230 via heat conduction and/or heat radiation between the respective second heating element 475 and the respective amplification zone 230. Each second heating element 475 is accommodated in the respective second recess portion 324 and is electrically connected to a common second power port 394 comprised in the support 300 (Fig. 1). The second power port 394 is selectively connectable to a power source (not shown). While in at least this example, each second heating element 475 comprises a copper trace, in alternative variations of this example each second heating element 475 can comprise at least one of: conductive trace serpentine; conducting paste. For example, the copper trace serpentine has a width of about 0.5mm, and adjacent traces are separated by a non-conducting gap of about 0.5mm.

Thus, in operation of the second heating system 470, each second heating element 475 generates heat, and applies the heat to the respective amplification zone 230, responsive to a second electric current being applied thereto, provided thereto by the power source via second port 394.

In at least this example, and referring again to Fig. 3, the device 10, and in particular the sample testing module 100, is further configured for enabling delivery of the aforesaid second quantity of transporting liquid to the preparation zone 210. In particular, the device 10 in general, and the sample testing module 100 in particular, is configured for enabling delivery of the aforesaid second quantity of transporting liquid to the preparation zone 210 in an automated manner that does not require manual intervention.

For this purpose, the device 10, and in particular the sample testing module 100, comprises a reservoir pad 500.

The reservoir pad 500, in at least this example, is formed from a sheet of fourth substrate material, configured for receiving therein the aforesaid second quantity of a transporting liquid. The support 300 is configured for supporting the reservoir pad 500 in longitudinal spaced relationship with respect to the reaction pad 200 by a gap 510. In particular, the reservoir pad 500 faces edge 212 of the preparation zone 210, wherein edge 212 is opposite edge 211 of the preparation zone 210.

In at least the embodiment illustrated in Figs. 1 to 4, the gap 510 can be devoid of any solid or semi-solid (for example porous) material mechanically connecting the reservoir pad 500 in longitudinal spaced relationship with respect to the reaction pad 200, and independent of the support 300. For example, such a gap may be entirely comprised of an air gap.

However, in alternative variations of this example, and referring for example to Figs. 8 and 9, the respective gap 510' can be traversed partially by a connecting strip 515' that spans the length of the gap and physically connects the reservoir pad 500 in longitudinal spaced relationship with respect to the reaction pad 200. Such a strip 515' typically has a thickness significantly less than the thickness T' of the respective reservoir pad 500' and/or of the respective reaction pad 200', to allow air to fill the remainder of the gap 510'. For example, such a connecting strip 515' may be in the form of or comprises a surface that is configured to render the gap hydrophobic - for example such a surface is a superhydrophobic surface (SHS).

For example, such a superhydrophobic surface was fabricated according to the following example. Polydimethylsiloxane (PDMS) (DOWSIL SYLGARD 184 Silicone Elastomer), was spin-coated (LAURELL WS-650SZ-6NPP/LITE) in 1:10 ratio, on a microfabricated silicon mold to obtain micro-structured surfaces. The product was let cure for 5hr at 60°C. The surfaces were composed of square micropillars (5pm X 5pm) organized in a square lattice with pillar spacing of 5, 10 and 15 pm. A nano structure Was next added to the pillars using chemical deposition of a 50mg of polyvinylchloride (PVC) (Sigma-Aldrich, polyvinyl chloride, high molecular weight, product number 81387), dissolved in a ethanol AR (Sigma-Aldrich St. Louis, MO), and tetrahydrofuran (THF) (Sigma-Aldrich St. Louis, MO) 1:1, 20ml mixture. In the deposition process, the solution was applied to the surfaces twice. After each time, the solvents were evaporated before brushing the deposited compound into the microstructure using a regular painting brush. The resulting surfaces were -125 pm thick and were cut to strips of 0.4cm X 1cm.

In at least this example, the reservoir pad 500 is made from a suitable porous fourth substrate material. For example, such a porous fourth substrate material can have a pore size ranging between about 0.01pm to about 10pm. Alternatively, the reservoir pad 500 can be made from any suitable fourth substrate material such as a suitable paper material. In yet some further embodiments, the reservoir pad 500 is made from chromatographic paper grade 4.

In at least this example, the support 300 is also configured for supporting the reservoir pad 500 in nominally flat and overlying relationship with respect to the support 300, and generally coplanar with the reaction pad 200. The support 300 comprises a second recess 350, of depth such as to ensure that an edge 512 of the reservoir pad 500 is facing at least a portion of the edge 212 of the reaction pad 210, via the gap 510.

In at least this example, the device 10, in particular the sample testing module 100, further comprises an electrically actuable microfluidic valve 700, having a normally closed configuration and an open configuration, for selectively enabling the second quantity of a transporting liquid to be transported to the reaction pad 200 from the reservoir pad 500 in an automated manner.

In the normally closed configuration, any such transporting liquid that is present in the reservoir pad 500 is prevented by the gap 510 from being transported to the preparation zone 210. Conversely, in the open configuration, any such transporting liquid that is present in the reservoir pad 500 is caused to be transported to the preparation zone 210 via the gap 510, responsive to a third electrical current being supplied to the electrically actuable microfluidic valve 700. The electrically actuable microfluidic valve 700 is electrically connectable to a suitable power supply (not shown) via third port 396 (Fig. 1).

Referring in particular to Fig. 4 and Fig. 6, in a first example of the electrically actuable microfluidic valve, the valve 700 comprises a positive electrode 725 and a negative electrode 727 spaced from one another in a longitudinal direction. The positive electrode 725 and the negative electrode 727 are located in the recess 350 and are configured for being in electrical contact with the transporting liquid in the reservoir pad 500, at least in operation of the device 10. The positive electrode 725 and the negative electrode 727 are electrically connected to a suitable electrical supply via third port 396. For example, such an electrical supply can be a battery, for example 30V battery. In at least this example, the negative electrode 727 is in close proximity to the gap 510, while the positive electrode 725 is further spaced from the gap 510.

Referring in particular to Fig. 1 and Fig. 5, the upper cover 950 comprises a first upper device portion 940 configured for being in overlying relationship with at least a first portion of the sample testing module 100, such a first portion including the preparation zone 210 and the reservoir pad 500. The upper cover 950, in particular the first upper device portion 940, is configured for fitting over the support 300 such that the gap 510 is hermetically sealed with respect to at least a portion of the reservoir pad 500 facing the gap 510 (including at least part of the edge 512), and with respect to at least a portion of the reaction pad 200 facing the gap (including at least a part of the preparation zone 210 including at least a part of the edge 212).

The first upper device portion 940 further comprises a transporting liquid portal 942, in registry with the reservoir pad 500, i.e., generally overlying the reservoir pad 500. The transporting liquid portal 942 is configured, at least in operation of the device, to enable insertion of the transporting liquid into the reservoir pad. In other words, the user can provide the transporting liquid to the reservoir pad 500 via the transporting liquid portal 942, and the transporting liquid remains in the reservoir pad 500 until such time as the device 10 is actuated, in particular until the electrically actuable microfluidic valve 700. In at least this example, the first upper device portion 940 further comprises a funnel arrangement 948 circumscribing the transporting liquid portal 942.

Without being bound by theory, inventors consider that, when applying a suitable voltage (for example 30V) between the positive electrode 725 and the negative electrode 727, the reservoir pad 500, wetted by the transporting liquid, operates in a similar manner to an electro-osmotic pump and urges the transporting liquid towards edge 512, and over the hermetically sealed gap 510, and into the preparation zone 210 via edge 212, thereby effectively operating as an active microfluidic valve.

Referring to Fig. 7, in a second example of the electrically actuable microfluidic valve, the valve 700 comprises a positive hydrophobic electrode 735 and a negative hydrophobic electrode 737, spaced from one another in a vertical direction. The positive electrode 735 is located in a bottom facing portion of the first upper device portion 940, while the negative electrode 737 is located in the recess 350 facing the positive electrode 735. A hydrophobic insulating layer 738 overlies the negative hydrophobic electrode 737. In this example, the upper cover 950, in particular the first upper device portion 940, is configured for fitting over the support 300 such that the gap 510 is hermetically sealed with respect to at least a portion of the reservoir pad 500 facing the gap 510 (including at least part of the edge 512), with respect to at least a portion of the reaction pad 200 facing the gap (including at least a part of the preparation zone 210 including at least a part of the edge 212), and also with respect to the positive electrode 735 on the upper side of the gap 510, and the hydrophobic insulating layer 738 on the lower side of the gap 510. Thus, each one of the positive electrode 735 and the negative electrode (in contact with the hydrophobic insulating layer 738) longitudinally spans at least a portion of the reaction pad 200 facing the gap (including at least a part of the preparation zone 210 including at least a part of the edge 212), the gap 510, and at least a portion of the reservoir pad 500 facing the gap 510 (including at least part of the edge 512). Thus, the positive electrode 735 and the hydrophobic insulating layer 738 are each configured for being in contact with the transporting liquid in the reservoir pad 500, at least in operation of the device 10. In other words, the positive hydrophobic electrode 735 is superposed on a first surface of the reaction pad spanning the gap 510 and at least a portion of the reaction pad facing the gap; the negative hydrophobic electrode 737 is overlaid with respect to the hydrophobic insulating layer 738; the negative hydrophobic electrode 737 and the hydrophobic insulating layer 738 being superposed on a second surface of the reaction pad, such that the hydrophobic insulating layer 738 is sandwiched between the second surface of the reaction pad 500 and the negative hydrophobic electrode 737, and spanning the gap 510 and at least a portion of the reaction pad facing the gap 510. The positive electrode 735 and the negative electrode 737 are electrically connected to a suitable electrical supply via third port 396. For example, such an electrical supply can be a battery, for example IkV battery. In at least this example, the hydrophobic insulating layer 738 comprises a plurality of through-holes 739. In this example, in operation the electrically actuable microfluidic valve is configured for generating a plasma discharge in the airspace provided in the gap 510 in response to a suitable third electrical current being provided to the electrically actuable microfluidic valve.

Without being bound by theory, inventors consider that, when applying a suitable voltage (for example 1KV) between the positive electrode 735 and the negative electrode 737, a localized plasma discharge is induced to ionize the surface of the hydrophobic insulating layer 738 that faces the gap 510, effectively providing a hydrophilic surface inducing capillary flow of the transporting liquid from the reservoir pad 500, over the hermetically sealed gap 510, and into the preparation zone 210 via edge 212, thereby effectively operating as an active microfluidic valve.

Referring again to Fig. 1 and Fig. 5, the upper cover 950, in particular the first upper device portion 940 further comprises a sample receiving portal 948 in registry with the preparation zone 210, i.e., generally overlying the preparation zone 210. The sample receiving portal 948 is configured, at least in operation of the device, to enable insertion of the sample (i.e., the sample to be tested) into the preparation zone 210.

In at least this example, and referring also to Fig. 1(a), the device 10 comprises an applicator member 920 having a graspable handle 922 and an enlarged base 924 having sample receiving zone 925 configured for enabling the sample to be delivered thereto from a user. The applicator member 920 is configured for being inserted through the sample receiving portal 948 into liquid contact with the preparation zone 210 to thereby enable the sample to be transferred from the sample receiving zone 925 to the preparation zone 210. For example, the sample can be applied to the sample receiving zone 925 from a patient by licking or spitting on the sample receiving zone 925.

In alternative variations of this example, the applicator member 920 can be omitted, and instead the sample can be directly inserted through the sample receiving portal 948 into liquid contact with the preparation zone 210 for example using a swab, pipette or other device, in which the user has previously deposited the sample. Alternatively, the user can deposit a sample directly through the sample receiving portal 948 into liquid contact with the preparation zone 210 directly from the mouth, for example.

Referring in particular to Fig. 1, the device 10 comprises a detection module 970 configured for enabling detecting of a detectable signal corresponding to a respective test parameter associated with said production of the at least one amplification product, in each of said plurality of amplification zones 230.

Referring also to Fig. 5, the upper cover 950 comprises a second upper device portion 960 configured for being in overlying relationship with at least a second portion of the sample testing module 100, such a second portion including the plurality of amplification zones 230. In at least this example, the detection module 970 is accommodated in the second upper device portion 960, and comprises a plurality of observation portals 972 formed in the second upper device portion 960. Each observation portal 972 is in registry with, i.e., overlies, a respective amplification zone 230, and at least in operation of the device 10 enables observation of a detectable signal corresponding to a respective test parameter associated with the respective amplification zone 230. Furthermore, the detection module 970 comprises a plurality of illumination sources (not shown) located in the underside of the second upper device portion 960. The illumination sources are configured for illuminating the plurality of amplification zones 230 at least in operation of the device 10, to thereby further facilitate visual observation of the amplification zones 230, in particular the color thereof.

In at least this example, the aforesaid detectable signal can be in the form of a color or a color change in the respective amplification zone 230, as disclosed in greater detail herein.

Also as disclosed in greater detail herein, in alternative variations of this example, the detection module 970 can be configured for enabling detecting of a detectable signal corresponding to a respective test parameter associated with said production of the at least one amplification product, wherein said test parameter is not necessarily a visually detectable color or color change. For example, such a test parameter can be a respective specific fluorescence parameter, and in such cases the detection module 970 can be configured for determining a respective detectable signal in the form of a fluorescence value of said respective specific fluorescence parameter. For example, the detection module 970 can comprises a fluorescence detection LED configured for measuring a detectable fluorescence signal in the form of a fluorescence value of said respective specific fluorescence parameter.

In another example, the test parameter can be a respective pH parameter, and the detection module 970 can be configured for determining a respective detectable signal in the form of a specific color (or a respective wavelength of said respective specific color) correlated to the pH.

In yet another example, the test parameter can be a respective pH parameter, and the detection module 970 can be configured for determining a respective pH value of the said respective specific pH parameter.

In yet another example, the test parameter can be a respective electrical resistance parameter, and the detection module 970 can be configured for detecting a respective electrical resistance value of said respective electrical resistance parameter. For example, the detection module 970 comprises sensing electrodes provided in the vicinity of the respective amplification zones; for example, two electrodes (for example two exposed metallic strips) can be placed outside each respective amplification zone, facing one another across the respective amplification zone, and each such pair of electrodes provides, directly or through a bypass filter, a suitable electrical signal to the detection module 970, corresponding to the magnitude of electrical resistance between the two electrodes via the respective amplification zone.

In operation of the device 10 for testing a sample, the reservoir pad 500 comprises the aforesaid second quantity of transporting liquid. This transporting liquid can be provided to the reservoir pad 500 via the transporting liquid portal 942.

It is to be noted that in alternative variations of this example, the device 10 is not configured for enabling automated delivery of the aforesaid second quantity of transporting liquid to the preparation zone 210, and instead such delivery is conducted in a manual manner by the user. Thus, the reservoir pad 500, the valve 700, can be omitted from the device 10 or the sample testing module 100, and the transporting liquid portal 942 can be omitted from the device 10. Instead, the aforesaid second quantity of transporting liquid can be directly delivered to the preparation zone 210 from outside the device 10, for example via a syringe or pipette.

In any case, and according to an aspect of the presently disclose subject matter, each one of said plurality of amplification zones 230 is configured for providing a respective amplification reaction, that in some non-limiting embodiments, are under isothermal conditions. Furthermore, each amplification reaction is a loop mediated isothermal amplification reaction (LAMP), wherein said each respective reaction mixture is a LAMP reaction mixture, and wherein each of said amplification zones comprises said respective LAMP reaction mixture (for example embedded within said respective substrate material of the respective amplification zone 230). In some particular and non-limiting embodiments, the amplification zone is configured for accommodating any appropriate reaction volume that comprises the reaction reagents and enables the amplification reaction. For example, a reaction volume and/or reagents volume of between about 250 to about Ipl. In mor specific embodiments, the reaction volume and/or reagents volume may be any one of 250pl or more, 200pl, 150pl, lOOpl, 95pl, 90pl, 85pl, 80pl, 75pl, 70pl, 65pl, 60pl, 55pl, 50pl, 45pl, 40pl, 35pl, 30pl, 25pl, 20pl, 15pl, lOpl, 9pl, 8pl, 7pl, 6pl, 5pl. 4pl, 3pl. 2 pl, Ipl, or less. In some specific embodiments, the amplification zone is configured for accommodating a reaction volume and/or reagents volume of about 7 l.

Thus, at least in use of the device 10, a first amplification zone 230 of said plurality of amplification zones 230 comprises a first said LAMP reaction mixture, said first amplification zone further comprises at least one set of primers specific for at least one nucleic acid sequence of said at least one nucleic acid sequence of interest. For example, the first said amplification zone 230 can come factory-ready with first said LAMP reaction mixture and said at least one set of primers; alternatively, the first said LAMP reaction mixture and at least one set of primers can be applied to the first amplification zone 230 at any suitable time prior to the test being conducted. Furthermore, at least in use of the device 10, a second amplification zone of said plurality of amplification zone 230 comprises a second said LAMP reaction mixture, and said second amplification zone 230 further comprises at least one set of primers specific for at least one nucleic acid sequence of at least one positive control. For example, the second said amplification zone 230 can come factory-ready with second said LAMP reaction mixture and said corresponding at least one set of primers; alternatively, the second said LAMP reaction mixture and the corresponding at least one set of primers can be applied to the second amplification zone 230 at any suitable time prior to the test being conducted.

Furthermore, at least in use of the device 10, a third amplification zone of said plurality of amplification zone 230 comprises a third said LAMP reaction mixture, said third amplification zone optionally further comprises at least one set of primers specific for at least one nucleic acid sequence of at least one negative control. For example, the third said amplification zone 230 can come factory-ready with third said LAMP reaction mixture and optionally said corresponding at least one set of primers; alternatively, the third said LAMP reaction mixture and optionally the corresponding at least one set of primers can be applied to the third amplification zone 230 at any suitable time prior to the test being conducted.

Furthermore, at least in use of the device 10, the sample preparation agents are comprised in the preparation zone 210, as discussed above.

Referring again to Fig. 1, the control module 900 in this example is affixed to the support 300, for example at lower face 304 of support 300. Thus, in this example, the control module 900 is integrated into the support 300 of said sample testing module 100.

In at least this example, the control module 900 comprises a microcomputer chip configured for carrying out an automated detection test, comprising the following steps: (a) responsive to initiation of the test by the user, allowing at least the sample to be tested and the first quantity of at least one sample preparation agent to interact with one another for a first predetermined time period; (b) following step (a), causing the first heating system 420 to heat the preparation zone 210 to the first temperature T1 for a first time duration;

(c) following step (b), operating the electrically actuable microfluidic valve 700 to cause the transporting liquid to be transported to the preparation zone 210 via the gap 510 and allowing the respective aliquot of each said prepared sample to be transported to the respective said amplification zone 230 under capillary action via the respective said branch element 220;

(d) following step (c), causing the second heating system 470 to heat each said amplification zone 230 to the second temperature T2 for a second time duration;

(e) following step (d), enabling detection of a detectable signal corresponding to a respective test parameter associated with said production of said at least one amplification product in each said amplification zone.

The control module 900 comprises a power input port 398 configured for enabling an electrical power connector to be connected thereto to enable electrical power from an external power source to be connected thereto, to thereby enable electrical power to be provided at least to said control module 900.

Optionally, the various power input ports 392, 394, 396, 398 can be consolidated into a single power input port 390.

For example, according to an aspect of the presently disclosed subject matter, such a sample can be a biological sample or an environmental sample.

Furthermore, in at least some examples the nucleic acid sequence of interest is a nucleic acid sequence of at least one pathogen. For example, said pathogen is a viral pathogen. For example, said viral pathogen is at least one corona virus (CoV), optionally, said CoV is Severe acute respiratory syndrome (SARS) CoV-2.

In an alternative variation of the example of Figs. 1 to 7, the device for enabling detection and monitoring of at least one nucleic acid sequence of interest in at least one sample is configured such that the control module 900 is not integrated with the support 300, but rather the control module 900 is separate from the support 300. This arrangement can enable the respective sample testing module to be configured as a stand-alone item capable of being selectively inserted and removed with respect to the device, in particular in a manner that precludes the possibility or probability of the device becoming contaminated with the sample. In this manner, the rest of the device 10 can be configured for multiple uses, each time with a different sample testing module.

For example, in such an example the device 10 comprises a testing chamber configured for enabling the sample testing module to be selectively inserted into the device 10 such as to be operatively coupled with the control module 900 and such as to enable the first heating system 420 and the second heating system 470 to be selectively provided with electrical power, and for enabling the respective sample testing module 200 to be selectively removed from the device 100 after the test is completed. In this example the device is configured for ensuring that the testing chamber avoids contamination from the sample.

According to an aspect of the presently disclosed subject matter, there is provided a system for enabling detection of at least one nucleic acid sequence of interest in one sample, for each one of a plurality of said samples. Such a system can comprise at least a corresponding plurality of said devices 10 as disclosed herein, and an electrical supply operatively connected to each said device 10.

Referring to Figs. 8 and 9, an alternative variation of the embodiment of Figs. 1 to 4, the respective device 10' corresponds to device 10 as disclosed herein with some differences, mutatis mutandis, as further clarified herein. Furthermore, the device 10' has similar dimensions to that of device 10 as disclosed herein, mutatis mutandis.

Furthermore, the device 10' can be operated in a similar manner to device 10 as disclosed herein, mutatis mutandis.

Thus, for example, device 10' comprises a sample testing module 100', a control module 900', an upper cover 950', detection module 970', and electrically actuable microfluidic valve 700', similar to sample testing module 100, a control module 900, an upper cover 950, detection module 970, and electrically actuable microfluidic valve 700 respectively, of device 10 as disclosed herein, mutatis mutandis, with some differences as will become clearer herein. For example, and as disclosed above, For example, the detection module 970' in at least this example comprises sensing electrodes provided in the vicinity of the respective amplification zones; for example, two electrodes 974' (for example two exposed metallic strips) can be placed outside each respective amplification zone, facing one another across the respective amplification zone, and each such pair of electrodes 974' provides, directly or through a bypass filter, a suitable electrical signal to the detection module 970', corresponding to the magnitude of electrical resistance between the two electrodes via the respective amplification zone. Furthermore, for example, the detection module 970' can comprises a fluorescence detection LED 973' configured for measuring a detectable fluorescence signal in the form of a fluorescence value of said respective specific fluorescence parameter. In some embodiments, the fluorescence detector may comprise LED as the exciting source and avalanche photodiode (APD) module as a photon sensor.

Furthermore, for example, sample testing module 100' comprises a reaction pad 200' and a support 300, similar to reaction pad 200 and a support 300 of sample testing module 100, as disclosed herein, mutatis mutandis, with some differences as will become clearer herein. In at least this example, the reaction pad 200' comprises a preparation zone 210' and a plurality of amplification zones 230', each amplification zone 230' being contiguous with the preparation zone 210' via at least one respective branch element 220, and a common branch element 235', similar to the reaction pad 200, preparation zone 210, amplification zones 230, branch elements 220, and common branch element 235 as disclosed herein mutatis mutandis. However, in at least this example, and as disclosed above, the respective preparation zone 210' is hexagonal, the respective amplification zones 230' are cruciform shaped, and the respective common branch 235' is trapezoidal, being integrally formed with the respective preparation zone 210'. Furthermore, the common branch 235' can be considered to form part of the preparation zone 210' in at least this example. Without being bound to theory inventors consider that the converging sides of the preparation zone 210' (including the common branch 235') in a direction towards the amplification zones 230' can facilitate or improve fluid flow towards the amplifications zones 230'. Furthermore, for example, the device 10' in particular the sample testing module 100', comprises reservoir pad 500' and gap 510', similar to reservoir pad 500' and gap 510' respectively, as disclosed herein, mutatis mutandis, with some differences as will become clearer herein. In at least this example, and as disclosed above, the respective gap 510' can be traversed partially by a connecting strip 515' that spans the length of the gap and physically connects the reservoir pad 500' in longitudinal spaced relationship with respect to the reaction pad 200'. Such a strip 515' typically has a thickness significantly less than the thickness T of the respective reservoir pad 500' and/or of the respective reaction pad 200', to allow air to fill the remainder of the gap 510'. For example, such a connecting strip 515' may be in the form of or comprises a surface that is configured to render the gap hydrophobic - for example such a surface is a superhydrophobic surface (SHS).

Furthermore, for example, support 300 comprises a first heating system 420' and a second heating system 470', similar to first heating system 420 and second heating system 470 respectively, as disclosed herein, mutatis mutandis. Furthermore, for example, support 300' comprises a first recess 320' and a second recess 350', similar to first recess 320 and second recess 350, respectively, as disclosed herein, mutatis mutandis, with some differences as will become clearer herein. In at least this example, support 300' comprises a gasket member 301', and the first recess 320' and the second recess 350' are formed in a gasket member 301' that is overlaid on the remainder of support 300'. The first recess 320' and the second recess 350' are sized and shaped in a complementary manner to the respective reaction pad 200' and reservoir pad 500' including gap 515'.

Furthermore, for example, the upper cover 950' comprises a first upper device portion 940' that comprises a transporting liquid portal 942', and a sample receiving portal 948', similar to first upper device portion 940, transporting liquid portal 942, and sample receiving portal 948 as disclosed herein, mutatis mutandis.

Furthermore, for example, the upper cover 950' comprises a second upper device portion 960' similar to second upper device portion 960, as disclosed herein, mutatis mutandis, with some differences as will become clearer herein. In at least this example, the observation portals 972 of the second upper device portion 960 of the example of Figs. 1 to 4 can be omitted in the example of Figs. 8, 9.

Furthermore, the device 10' further comprises a user interface 990', which can also be included in device 10, mutatis mutandis.

In a further aspect, the invention provides a method for the detection and monitoring of at least one target element in at least one sample. In more specific embodiments, the method of the invention comprises the following steps.

The first step (i), involves applying at least one sample in a preparation zone of a device as defined by the invention. The application of the sample in the preparation zone, initiates the automated detection test. However, it should be appreciated that in some alternative or additional embodiments, a more direct initiation by the user may be required, for example in cases that a sample is not placed in the device. Thus, in some alternative or additional embodiments, the initiation step may involve any one of the following steps, placing the applicator member in the sample receiving zone of the device of the invention (that is configured for enabling the sample to be delivered thereto), pressing a button, and/or turning a switch on to initiate the reaction. In some optional embodiment, this initial step may further comprise providing a second quantity of a transporting liquid to the reservoir pad of the device.

The second step (ii), allows for an automated detection test, controlled by the control module. In more specific embodiments, the control module controls the following steps or actions:

In the reaction start step (a), responsive to initiation of the test by the user, allowing at least the sample to be tested and the first quantity of at least one sample preparation agent to interact with one another for a first predetermined time period. It should be understood that the initiation of the test by the user may involve any direct or indirect signal/s or action/s, for example, any one of the following steps, adding the sample, placing the applicator member in the sample receiving zone of the device of the invention (that is configured for enabling the sample to be delivered thereto), adding a transporting liquid to the reservoir, pressing a button, and/or turning a switch on to initiate the reaction.

In the next step (b), following step (a), causing the first heating system to heat the preparation zone to the first temperature for a first time-duration. In the next step (c), following step (b), operating the electrically actuable microfluidic valve to cause the transporting liquid to be transported to the preparation zone via the gap and allowing the respective aliquot of each prepared sample to be transported to each respective reaction zone, under capillary action via the respective branch element.

In the next step (d), following step (c), causing the second heating system to heat each of the reaction zones of the plurality of reaction zones, to the second temperature for a second time duration to allow an reaction.

In the next step (e), following step (d), enabling detection of a detectable signal corresponding to a respective test parameter associated with the production of at least one reaction product by the reaction in each reaction zone.

The third step of the method of the invention (iii), involves determining the appearance of at least one test parameter associated with the production of the reaction product in each the reaction zones by detecting a detectable signal.

As indicated above, the first reaction zone comprises at least one set of primers specific for at least one nucleic acid sequence of the at least one nucleic acid sequence of interest. Therefore, the detection of a detectable signal in the first reaction zone of the plurality of reaction zones, indicates the presence of the nucleic acid sequence of interest in the sample.

In some embodiments, the target element detected and monitored by the disclosed method is at least one nucleic acid sequence of interest. Accordingly, the reaction performed by the method of the invention in the reaction zone is amplification reaction. Thus, the reaction zone/s may be referred to in some embodiments as amplification zone/s or amplification reaction zone/s.

Thus, the term "target element", that may be also referred to herein as a "target compound", or "target molecule" (used interchangeably), detected and/or monitored by the sample testing module, device, system and methods of the present disclosure, refer in some embodiments to any element or compound or molecule, either organic or synthetic. The target element or molecule may be in some embodiment a biological or non- biological entity. In certain embodiments of the method of the invention, the target molecule may be any target, for example: polypeptide, oligopeptide, peptide, nucleic acid, deoxyribonucleic acid, ribonucleic acid, glycoprotein, lipoprotein, glycolipoprotein, toxin, carbohydrate, polysaccharides, lipid, a small molecule, a small organic molecule and a non-organic molecule, peptidoglycans, hormones, receptors, antigens, antibodies, viruses, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, etc., without any limitation. In some specific embodiments, the target compound contaminating different substances, for example, environmental substances or water may be, but is not limited to, chemical contaminants, allergens, or drugs-of-abuse.

In yet some specific embodiments, the target element, target compound or target molecule, may be a target nucleic acid sequence of interest. The term "nucleic acid" is referred to often herein, and relates to DNA, RNA, single- stranded, partially singlestranded, partially double-stranded or double-stranded nucleic acid sequences; sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branch points and non-nucleotide residues, groups or bridges; synthetic RNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes; and any ribonucleotide, deoxyribonucleotide or chimeric counterpart thereof and/or corresponding complementary sequence and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8 -position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.

The terms "target nucleic acid sequence of interest", “nucleic acid sequence of interest”, "a target gene of interest", “a target gene", are used interchangeably, and refer in some embodiments to a nucleic acid sequence that may comprise or comprised within a gene or any fragment or derivative thereof. The target nucleic acid sequence or gene of interest may comprise coding or non-coding DNA regions, or any combination thereof. In some embodiments, the nucleic acid sequence of interest may comprise coding sequences and thus may comprise exons or fragments thereof that encode any product In other embodiments, the target nucleic acid sequence of interest may comprise non-coding sequences, as for example start codons, 5’ un-translated regions (5’ UTR), 3’ untranslated regions (3’ UTR), or other regulatory sequences, in particular regulatory sequences.

In some embodiments, the target gene or nucleic acid sequence of interest may be any nucleic acid sequence or gene or fragments thereof that display aberrant expression, stability, activity or function in a mammalian subject, as compared to normal and/or healthy subject. Such target gene or any fragments thereof or any target nucleic acid sequence may be in some embodiments, associated, linked or connected, directly or indirectly with at least one pathologic condition.

More specifically, the length of the nucleic acid sequence of interest may be about 100,000 nucleotides in length, or less than 75,000 nucleotides in length or less than 50,000 nucleotides in length, or less than 40,000 nucleotides in length, or less than 30,000 nucleotides in length, or less than 20,000 nucleotides in length, or less than 15,000 nucleotides in length, or less than 10,000 nucleotides in length, or less than 5000 nucleotides in length, or less than 1000 nucleotides in length, or less than 900 nucleotides in length, or less than 800 nucleotides in length, or less than 700 nucleotides in length, or less than 600 nucleotides in length, or less than 500 nucleotides in length, or less than 450 nucleotides in length, or less than 400 nucleotides in length, or less than 300 nucleotides in length, or less than 200 nucleotides in length, or less than 100 nucleotides in length, or less than 50 nucleotides in length, or less than 40 nucleotides in length, or less than 30 nucleotides in length, or less than 20 nucleotides in length, or less than 10 nucleotides in length.

As will be discussed in more detail herein after, in some particular embodiments, the target element, compound or molecule detected and/or monitored by the sample testing module, the device, the system and the methods of the present disclosure, may be a nucleic acid sequence of at least one pathogen. Thus, the present disclosure provides modules, devices, systems and methods for detecting and monitoring at least one pathogen, specifically, a viral pathogen. It should be understood that the method of the invention is a one step method that requires from the user only the initial step of initiating the automated test by placing the sample and/or the applicator member in the sample receiving zone of the device of the invention, pressing a button, or turning a switch on to initiate the automated test reaction. However, in some specific and non-limiting embodiments, the method may comprise an additional step of adding a transporting liquid either to the reservoir, or to the reaction chamber, performed by the user.

In some embodiments, the sample and the nucleic acid striping agents interact with one another for a first period of time, that may be in some embodiments, about 15 minutes in room temperature. In yet some further embodiments, immediately thereafter, the preparation zone is heated to a first temperature for a predetermined first length of time, specifically, for 5 minutes. In some embodiments, the first temperature is nominally about 95°C+/-5°C, thereby providing a prepared sample. Upon transferring of the prepared sample by the transporting liquid to said plurality of amplification zones, the amplification zones are heated to the second temperature for a predetermined second length of time sample. In some embodiments, the second temperature is nominally 65°C+/-5°C. In yet some further embodiments, the second length of time is about 35 minutes. These conditions allow an amplification reaction under isothermal conditions in each of said amplification zones. Upon completion of the amplification reaction, the produced amplification product is observed by the detection of a detectable signal.

Thus, the invention provides methods for detecting the presence of at last one pathogen in at least one sample. One of the initial steps of the methods of the invention involves the preparation of the sample that is placed in the sample preparation zone of the device of the invention. This step further comprises the incubation of the sample with a proteolysis reagent that can be in some embodiments at least one protease. In more specific embodiments, the sample preparation step, may involve incubation of the sample with the at least one protease or with any reagent comprising the protease, for a sufficient period of time under suitable conditions.

More specifically, in some embodiments the sample is incubated for about 10 to 20 minutes, specifically, about 10 or less, or, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more minutes, specifically, about 15 minutes at a temperature ranging between about 18 to 24 degrees Celsius, specifically, 18, 19, 20, 21, 22, 23, 24 degrees Celsius, more specifically, about 20 to 22 degrees Celsius, or more specifically, at room temperature. It should be noted that at this sample preparation step, the protease containing solution or buffer may further contain at least one further agent. Non-limiting examples for relevant additional agents may include any surfactant or detergent. For example, at least one polysorbatetype nonionic surfactant such as Polysorbate 20, also known as tween 20, and the like, at any suitable concentration. It should be understood that the invention encompasses the addition of any additional reagent or buffer required for the preparation step. As noted above, in some optional embodiments, the preparation step may further comprise incubation with at least one cheotropic agent. Thus, the protease solutions or buffers may comprise in some embodiments, at least one cheotropic agent.

Still further, subsequent to incubation with the nucleic acid stripping agent, for example, at least one protease, the method of the invention further involves the step of inactivation of the protease. Such step may be performed for example by heat inactivation. More specifically, by heating the sample up to about 95 degrees Celsius for a sufficient time period, specifically for about 2 to 10 minutes, more specifically, about 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, most specifically, for about 5 minutes. The prepared and inactivated sample is then transferred to the amplification zone that comprises a plurality of amplification zones.

In some embodiments, the second step of the method of the invention, specifically, the automated detection test that is controlled by the control module, may further comprise an additional preliminary step of sample preparation. According to such step, prior to step (a), the preparation zone, containing the sample, is preheated to said first temperature for a third time duration. This initial and optional step facilitates sample preparation, by leading to lysis of any proteins associated to the nucleic acid molecules in the sample. Such proteins may be part of biological membranes in case of a biological sample, for example, cellular membranes or viral envelops. This pre-heating step in some embodiments, may involved heating the preparation zone to a temperature ranging between about 50 to 80°C, specifically, 65°C+/-5°C, for thermal lysis.

To detect nucleic acid sequence specific for a particular nucleic acid sequence of interest in the sample, the next step of the methods of the invention involves amplification reaction. In some specific embodiments, such amplification is performed under isothermal conditions.

As used herein, the term "amplification" refers to increasing the number of copies of a nucleic acid molecule used as a template. Such nucleic acid molecule may comprise a gene, a fragment of a gene, or any control element or fragment thereof, for example, of at least one nucleic acid sequence of interest. In some embodiments, such sequence can be a nucleic acid sequence of a pathogen or of a positive or negative control.

As indicate above, the diagnostic method of the invention is based on amplification reaction to amplify the nucleic acid molecules in the sample. In some embodiments, the amplification reaction performed by the methods of the invention is an amplification reaction under isothermal conditions, optionally, a loop mediated isothermal amplification reaction (LAMP).

It should be noted that in some embodiments, all amplification zones in the device used by the methods of the invention comprise an amplification reaction mixture, specifically, LAMP amplification reaction mixture. In yet some further embodiments, at least one amplification zone of the plurality of amplification zones further comprise reagents specific for detection of the nucleic acid sequence of interest. By way of example, an amplification zone that comprise reagents specific for detecting the nucleic acid sequence of interest, may be referred to herein as a "first" amplification zone. In yet some further embodiments, the first amplification zone of the plurality of amplification zones comprises a LAMP reaction mixture, and further reagents for detection of the nucleic acid sequence of interest. Thus, in some specific embodiments, the first amplification zone further comprises at least one set of primers specific for at least one nucleic acid sequence of the at least one nucleic acid sequence of interest.

As will be discussed in more detail herein after, in some specific and non-limiting embodiments, such nucleic acid molecule may be any nucleic acid sequence of any gene or control element of a viral pathogen. In more specific embodiments, such pathogen is a viral pathogen. In yet some further embodiment, the nucleic acid sequence of interest detected by the disclosed methods may be at least a portion of SARS-COV2 nucleic acid molecule. In some particular and non-limiting embodiments of the invention, Gene N of SARS-CoV2 may be used as a template. The products of an amplification reaction are called amplification products. An example of in vitro amplification is the polymerase chain reaction (PCR), in which a sample (such as a biological sample from a subject, or any environmental sample) or any nucleic acid molecules obtained therefrom, is contacted with a pair of oligonucleotide primers, under conditions that allow for hybridization of the primers to a nucleic acid molecule in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid molecule. Examples of in vitro amplification techniques include real-time PCR, quantitative realtime PCR (qPCR), reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT- PCR); loop-mediated isothermal amplification; reverse-transcription LAMP (RT- LAMP); strand displacement amplification; transcription-mediated amplification, transcription-free isothermal amplification; repair chain reaction amplification; ligase chain reaction amplification; gap filling ligase chain reaction amplification; coupled ligase detection and PCR; and NASBA™ RNA transcription-free amplification.

In yet some further embodiments, the plurality of the amplification zones comprise in addition to the first amplification zone discussed above, also at least one amplification zone that comprise in addition to LAMP reaction mixture, also amplification reagents that are specific for at least one control nucleic acid sample. Such control reaction may be a positive or negative control, or both. By a way of example, the amplification zone that comprise these controls, may be referred to herein as a second and third amplification zones. Thus, in some embodiments, the device used by the methods of the invention may comprise at least one of:

(a), a second amplification zone that is designed to serve as a positive control, comprising a second LAMP reaction mixture. Such second amplification zone may therefore comprise in addition to LAMP reaction mixture, also at least one set of primers specific for at least one nucleic acid sequence of at least one positive control nucleic acid sequence; and

(b), a third amplification zone that is designed to serve as a negative control, comprising a third LAMP reaction mixture. Such third amplification zone may optionally comprise in addition to the third LAMP reaction mixture, also at least one set of primers specific for at least one nucleic acid sequence of at least one negative control nucleic acid sequence. In yet another option, a third amplification zone designed to serve as a negative control may comprise only a third LAMP reaction mixture with no primers.

As indicated above, the method of the invention comprises the step of performing an amplification reaction in the amplification zone of the device of the invention. Such amplification reaction uses at least one set of primers specific for the nucleic acid sequence of interest (e.g., of a pathogen) to be detected. However, in some embodiments, the method of the invention may further comprise controls that ensure that the reaction steps are appropriately performed. In some embodiments, the control may include performance of at least one additional control amplification reaction, performed in parallel or subsequently, with at least one additional aliquot of the sample. Thus, in some embodiments, the methods, kits, devices and systems of the invention encompass the use of control reaction mixtures, and even of control samples. The term "Control" refers to a reference standard, for example a positive control or negative control. A positive control is known to provide a positive test result. A negative control is known to provide a negative test result. However, the reference standard can be a theoretical or computed result, for example a result obtained in a population of reactions. Thus, a positive control may involve the use of primers specific for nucleic acid sequences known or expected to be present in the examined sample. For example, in case of a sample of a mammalian subject, specifically, a human subject, a positive control reaction mixture may comprise the use of primers specific for a gene of such mammal, for example, a human gene. In case of a sample obtained from a human source, any human gene may be used as a template for a suitable positive control. Non limiting example for nucleic acid sequence used as a positive control may be primers specific for the human POP7 gene that encodes the human ribonuclease P protein subunit p20 protein. Thus, according to such embodiments, a positive control amplification reaction may be performed using at least one set of primers specific for the POP7 gene. This amplification reaction is expected to produce a POP7 amplification product in case all steps of sample preparation and amplification reaction were performed appropriately. In such case, a negative result in the test amplification reaction performed using at least one set of primers specific the at least one pathogen, and a positive result (appearance of an amplification product) in the positive control reaction, indicates and ensures, at least partially, that nucleic acid sequences of the examined pathogen are indeed not present in the sample. Positive results (appearance of at least one amplification product) in both, the test and the positive control samples, may indicate that the nucleic acid sequence of the pathogen are present in the sample, thereby indicating the presence of the pathogen in the tested sample. It should be noted that the method of the invention may further use at least one negative control reaction that may be directed in some embodiments to nucleic acid sequences that are not expected to be present in the sample. Alternatively, a negative control reaction may comprise reaction reagents but may not include any primers. A negative control may be provided by a reaction mixture comprising primers for nucleic acid sequences that are irrelevant for the sample. Such negative control reactions ensures the specificity of the reagents, and the specificity of a positive result (appearance of an amplification product specific for the pathogen). Thus, a positive result of the test sample, a positive result of the positive control sample, and a negative result (no amplification product appears) in the negative control sample indicates the existence of the pathogen in the examined sample. Such control ensures a true positive result and minimize a false positive result.

Therefore, in some embodiments, the method of the invention may further comprise subjecting at least one aliquot of the prepared sample, for example, an aliquot transferred to the second and/or third amplification zones, to at least one amplification reaction using at least one set of primers specific for at least one control nucleic acid sequence. As indicated above, such control may be a positive control, or alternatively, or additionally, a negative control.

It should be however appreciated that the use of control samples, or at one least relative scale obtained from control samples may be also encompassed by the methods of the invention. As a control sample, the method of the invention may use a nucleic acid sequence of the searched pathogen or at least one sample known to comprise the pathogen. Alternatively, a control sample may include sample known to be negative for the specific searched pathogen or any nucleic acid sequence thereof. A further negative control may include in some embodiments, a control reaction in which no template has been added.

As indicated above, the reaction mixture contained within each reaction zone of the plurality of reaction, zones may comprise any reagent required for an amplification reaction. Typical amplification reactions contain primers (e.g., one, two, four, six or more primers), sample (which may or may not contain template to which the primers bind), nucleotides (corresponding to G, A, T and C), a buffering agent (ImM to 5mM Tris or 1.5mM to 5mM Tris or an equivalent buffer thereof), one or more salts (e.g., (Nt SCh, NaCl, MgSCh, MgCh, etc), a bacterial or archaebacterial polymerase (which may or may not be thermostable and may or may not have strand displacing activity), and any necessary cofactors and optional detergents, etc. Examples of thermostable polymerases that can be used for amplification, and specifically for in isothermal amplification reactions include, but are not limited to Taq, Tfi, Tzi, Tth, Pwo, Pfu, Q5®, Phusion®, OneTaq®, Vent®, DeepVent®, Klenow(exo-), Bst 2.0 and Bst 3.0 (New England Biolabs, Ipswich, MA), PyroPhage® (Lucigen, Middleton, Wl), Tin DNA polymerase, GspSSD LF DNA polymerase, Rsp (OptiGene, Horsham, UK) and phi29 polymerase, etc.

As noted above, the amplification reaction requires the use of primers, for example primers specific for nucleic acid sequences of the pathogen or for at least one control nucleic acid sequence.

Primers, as used herein, are short nucleic acids, generally DNA oligonucleotides 10 nucleotides or more in length (such as 10-60, 15-50, 20-40, 20-50, 25-50, or 30-60 nucleotides in length). Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs or sets of primers (such as 2, 3, 4, 5, 6, or more primers) can be used for amplification of a target nucleic acid, e.g., by PCR, LAMP, RT-LAMP, or other nucleic acid amplification methods known in the art. In some embodiments, where the pathogen to be detected is at least one viral pathogen, primers specific for a viral nucleic acid sequence are used. In some particular and non-limiting embodiments, where the pathogen to be detected is the SARS-CoV2 virus, primers specific for nucleic acid sequences of SARS-CoV2 virus, are used by the methods of the invention.

In some specific embodiments, where the Gene N of SARS-CoV2 is used as a template, suitable primers that may be used by the invention may comprise the nucleic acid sequence as denoted by any one of SEQ ID NO. 1, 2, 3, 4, 5 and 6. Still further, as indicated above, the initial step of the method of the invention involves the sample preparation step that may comprise contacting the sample with an effective amount of at least one nucleic acid stripping agent, for example, at least one protease, or any solution or mixture comprising the protease. In some embodiments such protease may be any serine protease.

As shown by the invention, at least one protease is used for sample preparation. A protease (also called a peptidase or proteinase) is an enzyme that catalyzes (increases the rate of) proteolysis, the breakdown of proteins into smaller polypeptides or single amino acids, by cleaving the peptide bonds within proteins by hydrolysis.

Thus, in some embodiments, proteases useful in the present invention may be at least one serine protease. Serine proteases (or serine endopeptidases) are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the (enzyme's) active site. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like. In some particular and nonlimiting embodiments, the protease used by the devices, systems, kits and methods of the invention for sample preparation may be proteinase K, or any variants, conjugates or derivatives thereof, or any solution, reagent, buffer or mixture thereof. Proteinase K (EC 3.4.21.64, protease K, endopeptidase K, Tritirachium alkaline proteinase, Tritirachium album serine proteinase, Tritirachium album proteinase K) is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Engyodontium album (formerly Tritirachium album). Proteinase K is able to digest hair (keratin), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8 (subtilisin). The molecular weight of Proteinase K is 28,900 daltons (28.9 kDa). Activated by calcium, the enzyme digests proteins preferentially after hydrophobic amino acids (aliphatic, aromatic and other hydrophobic amino acids). Although calcium ions do not affect the enzyme activity, they do contribute to its stability. Proteins will be completely digested if the incubation time is long and the protease concentration high enough. Upon removal of the calcium ions, the stability of the enzyme is reduced, but the proteolytic activity remains. Proteinase K has two binding sites for Ca2+, which are located close to the active center, but are not directly involved in the catalytic mechanism. The residual activity is sufficient to digest proteins, which usually contaminate nucleic acid preparations. Therefore, the digestion with Proteinase K for the purification of nucleic acids is usually performed in the presence of EDTA (inhibition of metal-ion dependent enzymes such as nucleases).

Proteinase K is also stable over a wide pH range (4—12), with a pH optimum of pH 8.0. An elevation of the reaction temperature from 37 °C to 50-60 °C may increase the activity several times, like the addition of 0.5-1% sodium dodecyl sulfate (SDS) or Guanidinium chloride (3 M), Guanidinium thiocyanate (1 M) and urea (4 M). The above-mentioned conditions enhance proteinase K activity by making its substrate cleavage sites more accessible. Temperatures above 65 °C, trichloroacetic acid (TCA) or the serine proteaseinhibitors AEBSF, PMSF or DFP inhibit the activity. Proteinase K will not be inhibited by Guanidinium chloride, Guanidinium thiocyanate, urea, Sarkosyl, Triton X-100, Tween 20, SDS, citrate, iodoacetic acid, EDTA or by other serine protease inhibitors like Na- Tosyl-Lys Chloromethyl Ketone (TLCK) and Na-Tosyl-Phe Chloromethyl Ketone (TPCK).

It should be understood that the proteinase K is n certain embodiments pat of the sample preparation reagents provided in the preparation zone of the disclosed device.

In some embodiments, either the sample preparation step, or the amplification reaction step, or even both steps, may comprise subjecting the sample to at least one cheotropic agent, that may be comprised in the preparation zone, in the plurality of the amplification zones, or in both. It means that either the sample preparation step, and/or the amplification reaction step may be performed in the presence of at least one cheotropic agent.

A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules (i.e. exerts chaotropic activity). This has an effect on the stability of the native state of other molecules in the solution, mainly macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect. For example, a chaotropic agent reduces the amount of order in the structure of a protein formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids, and may cause its denaturation.

Common chaotropic agents used include guanidinium chloride, n-butanol, ethanol, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. In some embodiments, the at least one chaotropic agent may be guanidine hydrochloride. Guanidinium chloride or guanidine hydrochloride, usually abbreviated GuHCl and sometimes GdnHCl or GdmCl, is the hydrochloride salt of guanidine.

Guanidinium chloride is a strong chaotrope and one of the strongest denaturants used in physiochemical studies of protein folding. It also has the ability to decrease enzyme activity and increase the solubility of hydrophobic molecules. At high concentrations of guanidinium chloride (e.g., 6 M), proteins lose their ordered structure, and they tend to become randomly coiled, i.e. they do not contain any residual structure.

In some embodiments, the chaotropic agent may be part of the regents included in the preparation zone and/or the amplification reaction zone of the disclosed device.

As noted above, the method of the invention comprises the step of amplification reaction to amplify either the pathogen nucleic acid sequence that may be present in the sample, or the control nucleic acid sequence (either positive control that is present in the sample, or a negative control that is not present in the sample). According to some embodiments, such steps are performed using isothermal conditions.

Isothermal amplification methods provide detection of a nucleic acid target sequence in a streamlined, exponential manner, and are not limited by the constraint of thermal cycling. Although these methods can vary considerably, they all share some features in common. For example, because the DNA strands are not heat denatured, all isothermal methods rely on an alternative approach to enable primer binding and initiation of the amplification reaction: a polymerase with strand-displacement activity. Once the reaction is initiated, the polymerase must also separate the strand that is still annealed to the sequence of interest. Isothermal amplification reactions are performed using constant thermal conditions, specifically, either moderate temperature reactions (ranges between about 25 to about 40°C) or higher temperature (ranges between about 50 to about 65°C).

Still further, isothermal methods typically employ unique DNA polymerases for separating duplex DNA. DNA polymerases with this ability include any Klenow exopolymerase, specifically, any Klenow Fragment (3'— > 5' exo-) that is an N-terminal truncation of DNA Polymerase I which retains polymerase activity, but has lost the 5'— 3' exonuclease activity. In some non-limiting examples, Klenow exo- polymerases applicable in the present disclosure include the Bsu Klenow exo-, large fragment, the phi29 for moderate temperature reactions (25-40°C), and the large fragment of Bst DNA polymerase for higher temperature (50-65 °C) reactions. In some embodiments, the amplification reaction performed under isothermal conditions may be at least one of Loop-Mediated Isothermal Amplification (LAMP) that may be performed at 65°C, Strand Displacement Amplification (SDA), that may be performed at 60°C, Helicase-Dependent Amplification (HDA), that may be performed at 65°C, Recombinase Polymerase Amplification (RPA), that may be performed at 37°C, and Nucleic Acid Sequences Based Amplification (NASBA), that may be performed at 40- 55°C.

More specifically, LAMP uses 4 to 6 primers recognizing 6 to 8 distinct regions of target DNA. A strand-displacing DNA polymerase initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification. LAMP is rapid, sensitive, and amplification is so extensive that the magnesium pyrophosphate produced during the reaction can be seen by eye, making LAMP well-suited for field diagnostics.

SDA, or a similar approach, Nicking Enzyme Amplification Reaction (NEAR), relies on a strand-displacing DNA polymerase, typically Bst DNA Polymerase, Large Fragment or Klenow Fragment (3 ’-5’ exo-), to initiate at nicks created by a strand-limited restriction endonuclease or nicking enzyme at a site contained in a primer. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification. NEAR is extremely rapid and sensitive, enabling detection of small target amounts in minutes. SDA and NEAR are typically utilized in clinical and biosafety applications.

HDA employs the double-stranded DNA unwinding activity of a helicase to separate strands, enabling primer annealing and extension by a strand-displacing DNA polymerase. Like PCR, this system requires only two primers. HDA has been employed in several diagnostic devices and FDA- approved tests.

RPA uses a recombinase enzyme to help primers invade double-stranded DNA. T4 UvsX, UvsY, and a single stranded binding protein T4 gp32 form D-loop recombination structures that initiate amplification by a strand-displacing DNA polymerase. RPA is typically performed at ~37 °C and, unlike other methods, can produce discrete amplicons up to 1 kb.

Still further, NASBA and Transcription Mediated Amplification (TMA) are both isothermal amplification methods that proceed through RNA. Primers are designed to target a region of interest; one of the primers must include the promoter sequence for T7 RNA polymerase at the 5’ end. NASBA and TMA reactions are utilized in a range of clinical diagnostics.

In some embodiments, the amplification step used by the disclosed methods is a loop mediated isothermal amplification reaction (LAMP).

More specifically, Loop-mediated isothermal amplification (LAMP) refers to a method for amplifying nucleic acid. The method is a single-step amplification reaction utilizing a DNA polymerase with strand displacement activity (e.g., Notomi et al., Nucl. Acids. Res. 28:E63, 2000), utilizing 4-6 primers designed to amplify the gene target through creation of stem-loop structures that aid in synthesizing new DNA by the polymerase. Synthesis of DNA occurs rapidly at a constant temperature, unlike PCR which requires specialized equipment for temperature cycling. LAMP is also highly specific, since several primers are used to amplify a specific nucleic acid sequence. More specifically, at least four primers, which are specific for eight regions within a target nucleic acid sequence, are typically used for LAMP; however, in some embodiments, two primers may be used for LAMP. The primers may include in some embodiments a forward outer primer (F3), a backward outer primer (B3), a forward inner primer (FIP), and a backward inner primer (BIP). A forward loop primer (Loop F or LF), and/or a backward loop primer (Loop B or LB) can also be included in some embodiments. The amplification reaction produces a stem- loop DNA with inverted repeats of the target nucleic acid sequence. To amplify RNA sequences using LAMP, reverse transcriptase (RT) is added to the reaction. This variation is referred to as RT-LAMP. In contrast to PCR, LAMP and RT-LAMP are carried out at a constant temperature and do not require a thermal cycler. Thus, in some embodiments, either one set of primers or a multiplex of several sets of primers may be used.

It should be noted that the production of an amplification product indicates the existence of the specific nucleic acid sequence (either of the pathogen, or the control) may be detected by any parameter, for example, change in pH, change in conductivity, change in electric charge, change in ATP production reflecting PPi production by the polymerase, intercalation of products, or change in pyrophosphate precipitates. In some embodiments, the change in pH may be detected using any pH sensitive indicator. Thus, in general, in one aspect, an amplification reaction mix is provided, in some embodiments dried and/or embedded within the reaction zone, that includes: a polymerase, 1.5 mM to 5 mM Tris or an equivalent thereof, a single pH sensitive dye, dNTP, one or more primers; and one or more templates wherein the reaction mix changes color only during amplification reaction. In embodiments of the method, change in color of pH sensitive dyes resulting from amplification is observed in any buffer at a concentration equivalent to 1.5mM to 5mM Tris such as 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM or 5mM Tris or any other concentration in the specified range. A person of ordinary skill in the art will be acquainted with Tris buffer and equivalent buffers that are standard within the art such as Tricine, Bicine, TAPS, MOBS, and Hepes (Sigma Aldrich, St. Louis, MO) including DIPSO, TAPSO, HEPPSO, POPSO, TEA, EPPS, HEPBS, AMPD and TABS (Sigma Aldrich, St. Louis, MO).

In yet some alternative embodiments, the LAMP reaction is performed using a reaction mixture comprising at least one detectable compound capable of intercalating into the a double strand DNA, thereby providing a detectable signal upon production of at least one amplification product. In some embodiments, the detectable compound may be detected either directly or indirectly. In yet some further embodiments, the detectable compound may be a fluorescent compound. In more specific embodiments, such fluorescent compound may be SYBER green. More specifically, SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain that binds to DNA. The resulting DNA-dye-complex absorbs best 497 nanometer blue light (Ln» = 497 nm) and emits green light (Lnax = 520 nm). The stain preferentially binds to double-stranded DNA, but will stain single-stranded (ss) DNA with lower performance. SYBR Green can also stain RNA with a lower performance than ssDNA.

In yet some alternative embodiments, for detecting and/or quantitating the amplification product, EdU (5-Ethynyl-2'-deoxyuridine) that is capable of intercalating to the nucleic acid sequence, is used by the methods of the invention. More specifically, EdU is a thymidine analog that can be incorporated into replicating DNA. The alkyne handler can be used for subsequent ligation to azide-containing molecules through a highly efficient click chemistry reaction. In some embodiments the LAMP reaction is performed using a reaction mixture comprising a pH sensitive indicator dye providing a detectable signal upon production of at least one amplification product.

Thus, in some embodiments, a pH sensitive indicator dye is used by the invention to provide a detectable signal upon production of at least one amplification product. A pH- sensitive indicator dye is a dye that undergoes a color change as pH increases or decreases. In the context of the present disclosure, pH-sensitive indicator dyes can be used to detect nucleic acid amplification products. When a DNA polymerase incorporates a deoxynucleoside triphosphate into a nascent DNA, the released by-products include a pyrophosphate moiety and a hydrogen ion. Thus, as nucleic acid amplification products increase in a reaction, the pH of the solution decreases, resulting in a color change of the pH-sensitive dye. In some embodiments, the pH sensitive indicator dye is a colored dye detectable in visible light. In some embodiments, the change in color of the amplification reaction may be visible by eye. As such, the detecting step may be done by a naked eye. The detection may be qualitative or quantitative. In some embodiments, for example, the method may comprise simply detecting whether there has been a change in the color during the amplification reaction, thereby indicating the presence or absence of an amplification product. In some embodiments, the color of the reaction may be compared to a color chart or the color of one or more controls, e.g., an amplification reaction that does not contain any template and/or an amplification reaction that contains a different template that is amplified in the amplification reaction, thereby allowing a user to determine if the reaction contains a product or not. In other embodiments, for example, the method may comprise quantifying the change in the color of the amplification reaction, thereby indicating the amount of product in the amplification product. In some embodiments, the color of the reaction may be compared to a color chart or the color of one or more controls, e.g., amplification reactions that contain varying amounts of a different template that is amplified in the amplification reaction, thereby allowing a user to quantify the amount of product and/or template in the amplification reaction. In some cases, the color of the reaction may be read within 1 hour, e.g., 5 minutes -50 minutes, after the reaction starts.

The pH-sensitive dye used may be selected according to any characteristic, e.g., their color change (i.e., whether they change from violet to yellow, red to yellow, or yellow to red, etc.), the initial pH of the amplification reaction (e.g., whether the reaction initially has a pH of greater than pH 8.0, a pH of 7.5-8.5, or a pH of 6.5-7.5, etc., and whether the color change is going to be detected using a machine (e.g., a colorimeter, fluorimeter or spectrophotometer) by human eye (i.e., without the aid of a colorimeter, fluorimeter or spectrophotometer). The selected dye generally changes color in a pH range at which the polymerase is operational (e.g., a pH of 5-10).

There are a wide range of pH-sensitive dyes that can be used in the present method. Examples of pH sensitive dyes that change color at different pH values are described below. These examples are not intended to be limiting. Suitable visible dyes include: neutral red, which has a clear-yellow color when the pH is higher than 8 and a red color when the pH is less than 6.8; phenol red, which has a red color when the pH is higher than 8 and a yellow color when the pH is less than 6.4; cresol red, which has a reddish- purple color when the pH is higher than 8.8 and a yellow color when the pH is less than 7.2; thymol blue, which has a blue color when the pH is higher than 9.6 and a yellow color when the pH is less than 8.0; phenolphthalein, which has a fuchsia color when the pH is higher than 10 and colorless when the pH is less than 8.3; and naphtholphthalein, which has a greenish color when the pH is higher than 8.7 and a pale -reddish color when the pH is less than 7.3. The properties of some of the dyes that can be used in the method are summarized in Table 1. The term "visual" includes those dyes that can be detected by the "naked eye" where the "naked eye" refers to visualization without instrumentation. The "naked eye" includes the use of contact lenses and/or spectacles. The contact lenses/spectacles may include lenses and/or tinting to enhance or eliminate certain wavelengths of light.

Other examples of pH indicators include: methyl yellow, methyl orange, bromophenol blue, naphthyl red, bromocresol green, methyl red, azolitmin, nile blue, thymolphthalein, alizarin yellow, salicyl yellow, nitramine, phenol red, cresol red, neutral red, m-cresol purple, bromocresol purple, naphtholphthalein, thymol blue and naphtolphthalein. The color may transition outside the range of traditional DNA polymerase tolerances, but the principle of amplification detection may be applied to alternate detection methods with an indicator appropriate for desired pH range. pH-sensitive fluorescent dyes can be detected using a fluorometer. Like visual dyes mentioned above, pH-sensitive fluorescence dyes have different levels of fluorescence emission or a shift of peak emission wavelength at different pH. Both the change in brightness and the shift in peak absorption can be easily detected using systems that are equipped with proper filter sets.

It should be understood that the result of the reaction can be either assessed by eye or by a colorimetric measure such as an ODmeter, and the like.

In yet some alternative embodiments, the pH sensitive indicator dye is a fluorescent indicator dye. Fluorescent indicator dye refers to a fluorescent compound that responds to changes in environmental conditions (such as pH or metal ion concentration) by changes in fluorescence properties. In some examples, fluorescence of a fluorescent indicator dye is increased by a change in pH. The fluorescent indicator dye can be detected by any suitable method, including visually (e.g., under ambient or ultraviolet light) or using instrumentation for detection of fluorescence (such as a fluorimeter or realtime PCR system). Exemplary fluorescent indicator dyes include, for example, calcein, hydroxynaphthol blue, Mag-Fura-2 and Magnesium Green (Life Technologies, Grand Island, NY) and Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, and Asante Magnesium Green (TEF Labs, Austin, TX). Other fluorescent pH indicators include 2',7'-bis-(2-carboxyethyl)-5- (and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Life Technologies, Grand Island, NY) which at pH 9 has a absorbance/ emission profile of A _ma x 500 nm/Em _m ax535 nm, 5-(and-6) carboxy SNARF-1 which features a shift in fluorescence based on pH. At high pH (pH 9) SNARF-1 maximum absorbance/emission at A _ma x 575 nm/ Em _m ax 650nm.

In some examples, fluorescence from the fluorescent indicator dyes useful in the methods disclosed herein is visibly detectable (for example, by eye, such as a colorimetric reagent), while in other examples, the fluorescence is detectable using an instrument, such as a fluorimeter.

Visual and fluorescent dyes including those mentioned above can be chemically modified to have altered colorimetric properties in response to pH changes. These modifications can create dyes that are either brighter or change color at a narrower pH range and thus allow a better detection.

In some embodiments, where a pH sensitive dye is not comprised within the reaction mixture, the change of pH as a function of the production of the amplification product, may be measured either manually or mechanically (pH meter etc.). In yet some further alternative embodiments, the presence of an amplification product may be detected by measuring the change in other parameters, for example, electric charge or conductivity or electrical resistance of the sample after amplification reaction. In some embodiments, change in electric charge or in electrical resistance measured or detected by the methods of the invention may be caused by release of hydrogen and production of a hydrogen potential. More specifically, the change in electric charge is detected and/or measured by the methods of the invention, using for example a membrane that enables accumulation of protons. Electricity would be measured in the chamber in which the accumulation of the protons is enabled, by any suitable means. In some embodiments, change in electrical resistance measured or detected by the methods of the invention may be determined via sensing electrodes provided in the vicinity of the respective amplification zones; for example, two electrodes can be placed outside each respective amplification zone, facing one another across the respective amplification zone.

In yet some further embodiments, the detection of DNA polymerase activity that reflects the production of the amplification product in the reaction chamber may be assessed in some embodiments by measuring the production of inorganic pyrophosphate (PPi). In some particular and non-limiting embodiments, such PPi may be assessed by measuring the production of ATP. More specifically, in some embodiments, by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA). The PPi formed in the DNA polymerase reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase.

In yet some further embodiments, the production of the amplification product may be performed in some embodiments by measuring and/or detecting inorganic pyrophosphate (PPi) that reflects DNA polymerase activity. Precipitates of the pyrophosphate with Calcium, may be measured, by any suitable means, for example, using in a spectrophotometer.

It should be further understood that in some embodiments, the amplification reaction reagents as discussed are provided and/or embedded into the amplification zone of the disclosed device. As indicated above, the detection means for detecting an amplification product produced in the sample tested by the methods, kits, systems and devices of the invention can be a fluorophore, a radiolabel, or any other signal-emitting, signal inducing, or otherwise chemically or physically discernible. The various detection systems are well known to those skilled in the art.

The term “detectable” as used herein refers to the presence of a detectable signal generated from a detectable chemical reaction that is immediately detectable by observation, instrumentation, or film. The term “detectable signal” (also referred to by some aspects of the invention as a test parameter) as used herein refers to any compound or means that leads to occurrence of, or a change in, a signal that is directly or indirectly detectable (observable) either by visual observation or by instrumentation, thereby reflecting the production of amplification product in accordance with the invention. Typically, the detectable signal is detectable in an optical property (“optically detectable”) as reflected by a change in the wavelength distribution patterns, or intensity of absorbance, or a combination of such parameters.

The method of the invention enables detection of at least one nucleic acid sequence of interest, for example, a nucleic acid sequence of a pathogen, and specifically the presence of at least one nucleic acid sequence of at least one pathogen in any sample. Thus, in some embodiments, the devices, systems, and methods of the invention may be suitable for detecting at least one pathogen, and specifically, nucleic acid molecules thereof, in any surface, substance or any sample thereof.

In yet some further embodiments, the sample examined by the methods of the invention is at least one of a biological sample and an environmental sample.

The terms "sample", "test sample" and "specimen" are used interchangeably in the present specification and claims and are used in its broadest sense. They are meant to include both biological and environmental samples and may include an exemplar of synthetic origin. This term refers to any media that may contain the pathogen and may include fluid, cell and/or tissue samples. In some embodiments herein, the biological sample is a fluid sample. Fluid sample include, but are not limited to, saliva, mucosa, feces, serum, urine, blood, plasma, cerebral spinal fluid (CSF), milk, bronchoalveolar lavage (BAL) fluid, rinse fluid obtained from wash of body cavities, phlegm, pus. Still further, biological samples including samples taken from various body regions (nose, throat, vagina, ear, eye, skin, sores), food products (both solids and fluids) and swabs taken from medicinal instruments, apparatus, materials), samples from various surfaces [hospitals, elderly homes, food manufacturing facilities, slaughter houses pharmaceutical equipment (catheters etc), food preparation or packaging products), solutions and buffers], sewage etc. In some embodiments, the sample is at least one of a biological sample and an environmental sample.

Biological samples may be provided from animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products, food designed for human consumption, a sample including food designed for animal consumption, food matrices and ingredients such as dairy items, vegetables, meat and meat by-products, waste and sewage. In some embodiments, biological samples may include saliva, mucosa (nasal or oral swab samples), feces, serum, blood, urine, anterior nares (nasal swab) specimen collected by a healthcare professional or by onsite or home self-collection (using a flocked or spun polyester swab Nasopharyngeal (NP) specimens throat swab. Biological samples and specimens may be obtained from human as well as from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, birds, fish, lagamorphs, rodents, etc.

As indicated herein before, the present invention provides methods, kits, devices and systems that may be applicable for detecting at least one pathogen in any sample, including environmental samples. More specifically, environmental samples include environmental material such as surface matter, earth, soil, water, air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention. The sample may be any media, specifically, a liquid media that may contain the pathogen. Typically, substances, surfaces and samples or specimens that are a priori not liquid may be contacted with a liquid media which is used and tested by the methods, kits, devices and systems of the invention of the invention.

In some embodiments, the methods, kits, devices and systems of the invention may be applicable for detecting at least one pathogen in food or food products and beverages. More specifically, by the term “food”, it is referred to any substance consumed, usually of plant or animal origin. Some non limiting examples of animals used for feeding are cows, pigs, poultry, etc. The term food also comprises products derived from animals, such as, but not limited to, milk and food products derived from milk, eggs, meat, etc. A drink or beverage is a liquid which is specifically prepared for human consumption. Non limiting examples of drinks include, but are not limited to water, milk, alcoholic and nonalcoholic beverages, soft drinks, fruit extracts, etc.

In yet some further embodiments, the nucleic acid sequence of interest may be any nucleic acid sequence of any biological entity. In some embodiments, the nucleic acid sequence of interest is a nucleic acid sequence of at least one pathogen. Thus, according to some embodiments, the method of the invention may be useful for the detection and monitoring of at least one pathogen in at least one sample.

Thus, in some embodiments, the method of the invention provides the detection of any pathogen in any sample. The present invention provides devices, systems, kits and methods for detecting pathogens in a sample. As used herein, the term “pathogen” refers to an infectious agent that causes a disease in a subject host. Pathogenic agents include prokaryotic microorganisms, lower eukaryotic microorganisms, complex eukaryotic organisms, viruses, fungi, mycoplasma, prions, parasites, for example, a parasitic protozoan, yeasts or a nematode.

In yet some further embodiments, the methods of the invention may be applicable for detecting a pathogen that may be in further specific embodiment, a viral pathogen or a virus. In some embodiments, the pathogen may be at least one viral pathogen.

The term "virus" as used herein, refers to obligate intracellular parasites of living but non-cellular nature, consisting of DNA or RNA and a protein coat. Viruses range in diameter from about 20 to about 300 nm. Class I viruses (Baltimore classification) have a double-stranded DNA as their genome; Class II viruses have a single-stranded DNA as their genome; Class III viruses have a double-stranded RNA as their genome; Class IV viruses have a positive single-stranded RNA as their genome, the genome itself acting as mRNA; Class V viruses have a negative single-stranded RNA as their genome used as a template for mRNA synthesis; and Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. It should be noted that the term “viruses” is used in its broadest sense to include any virus, specifically, any enveloped virus. In some specific embodiments, the viral pathogen may be of any of the following orders, specifically, Herpesvirales (large eukaryotic dsDNA viruses), Ligamenvirales (linear, dsDNA (group I) archaean viruses), Monone gavirales (include nonsegmented (-) strand ssRNA (Group V) plant and animal viruses), Nidovirales (composed of (+) strand ssRNA (Group IV) viruses), Ortervirales (singlestranded RNA and DNA viruses that replicate through a DNA intermediate (Groups VI and VII)), Picornavirales (small (+) strand ssRNA viruses that infect a variety of plant, insect and animal hosts), Tymovirales (monopartite (+) ssRNA viruses), Bunyavirales contain tripartite (-) ssRNA viruses (Group V) and Caudovirales (tailed dsDNA (group I) bacteriophages).

In some embodiments, the viral pathogens of the invention may be DNA viruses, specifically, any virus of the following families: the Adenoviridae family, the Papovaviridae family, the Parvoviridae family, the Herpesviridae family, the Poxviridae family, the Hepadnaviridae family and the Anelloviridae family.

In yet some further specific embodiments, the viral pathogens of the invention may be RNA viruses, specifically, any virus of the following families: the Reoviridae family, Picornaviridae family, Caliciviridae family, Togaviridae family, Arenaviridae family, Flaviviridae family, Orthomyxoviridae family, Paramyxoviridae family, Bunyaviridae family, Rhabdoviridae family, Filoviridae family, Coronaviridae family, Astroviridae family, Bornaviridae family, Arteriviridae family, Hepeviridae family and the Retroviridae family. Of particular interest are viruses of the families adenoviruses, papovaviruses, herpesviruses: simplex, varicella-zoster, Epstein-Barr (EBV), Cytomegalo virus (CMV), pox viruses: smallpox, vaccinia, hepatitis B (HBV), rhinoviruses, hepatitis A (HBA), poliovirus, respiratory syncytial virus (RSV), Middle East Respiratory Syndrome (MERS-CoV), Severe acute respiratory syndrome (SARS- Cov), SARS-CoV2, corona virus, rubella virus, hepatitis C (HBC), arboviruses, rabies virus, influenza viruses A and B, measles virus, mumps virus, human deficiency virus (HIV), HTLV I and II and Zika virus.

In some specific and embodiments, the devices, systems, and methods of the invention may be suitable for detecting at least one corona virus (CoV). CoVs are common in humans and usually cause mild to moderate upper-respiratory tract illnesses. There are four main sub-groupings of coronaviruses, known as alpha, beta, gamma, and delta. The seven coronaviruses known to-date as infecting humans are: alpha coronaviruses 229E and NL63, and beta coronaviruses OC43, HKU1, SARS-CoV and SARS-CoV2, and MERS-CoV (the coronavirus that causes Middle East Respiratory Syndrome, or MERS). The SARS-CoV and SARS-CoV2 are a lineage B beta Coronavirus and the MERS-CoV is a lineage C beta Coronavirus.

Coronaviruses are species in the genera of virus belonging to one of two subfamilies Coronavirinae and Torovirinae in the family Coronaviridae, in the order Nidovirales. Herein this term refers to the entire family of Coronavirinae (in the order Nidovirales). Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and with a nucleocapsid of helical symmetry. The genomic size of coronaviruses ranges from approximately 26 to 32 kilobases, the largest for an RNA virus. The name "coronavirus" is derived from the Latin corona, meaning crown or halo, and refers to the characteristic appearance of virions under electron microscopy (E.M.) with a fringe of large surface projections creating an image reminiscent of a crown. This morphology is created by the viral spike (S) peplomers, which are proteins that populate the surface of the virus and determine host tropism.

There are many CoVs that naturally infect animals, the majority of these usually infect only one animal species or, at most, a small number of closely related species, but not humans. CoV strains that are particular subject of the present invention, due to their extreme virulence and hazard in humans, are those that have been transmitted from animals to humans, specifically the SARS-CoV2. Thus the term Coronaviruses (designated herein CoVs) for the purposes of the present invention encompasses four main sub-groupings of coronaviruses, known as Alpha, Beta, Gamma, and Delta. More specifically, under this term is meant the enveloped viruses with a positive-sense RNA genome (ssRNA+) and with a nucleocapsid of helical symmetry; and also large RNA viruses with the genomic size of ranges from approximately 26 to 32 kilobases; and further viruses with the characteristic morphology of large, bulbous surface projections under electron microscopy, which is created by the viral spike (S) peplomers, i.e. viral surface proteins determining host tropism and immunogenicity.

In some non-limiting embodiments, the present invention may be particularly applicable to the seven CoVs that can infect humans, specifically the Alpha CoVs 229E and NL63, and Beta CoVs OC43, HKU1, SARS-CoV, SARS-Cov2 and MERS-CoV. Specifically, for the Beta-CoVs, which are of the greatest clinical importance concerning humans, these are OC43, and HKU1 of the A lineage, SARS-CoV of the B lineage, and MERS- CoV of the C lineage. MERS-CoV is the first Beta CoV belonging to lineage C that is known to infect humans. The Alpha and Beta CoVs genera descend from the bat gene pool. Coronaviruses infecting animals are also contemplated, in particular the bat coronaviruses HKU4 and HKU5.

Within the above group of human CoVs, of particular relevance to the present invention are the SARS-CoV2 associated with COVID 19, S ARS CoV associated with Severe Acute Respiratory Syndrome, and the MERS CoV associated with Middle East Respiratory Syndrome, as for being the primary causes of life-threatening infectious diseases and epidemics in humans.

The other human CoVs are believed to cause a significant percentage of all common colds in human adults (primarily in the winter and early spring seasons). In certain individuals CoVs may further be a direct or indirect cause of pneumonia, i.e. direct viral pneumonia or a secondary bacterial pneumonia.

Still further, in some specific embodiments, the method of the invention may be particularly applicable in detecting the Severe acute respiratory syndrome (SARS) CoV-2, and any mutants or variants thereof.

In yet some more specific embodiments, the viral pathogen is at least one CoV. In some particular embodiments, such CoV may be Severe acute respiratory syndrome (SARS) CoV-2. Thus, in accordance with some embodiments, the invention provides methods for the detection and monitoring of SARS CoV-2 in at least one sample.

Thus, of particular relevance to the present invention is the SARS CoV2 associated with Severe Acute Respiratory Syndrome 2, as for being the primary cause of life-threatening infectious diseases and epidemics/pandemics in humans referred to as COVID 19 (Coronavirus Disease 2019).

SARS CoV2 is a member of the subgenus Sarbecovirus (beta-CoV lineage B). Its RNA sequence is approximately 30,000 bases in length. SARS-CoV-2 is unique among known betacoronaviruses in its incorporation of a polybasic cleavage site, a characteristic known to increase pathogenicity and transmissibility in other viruses.

Each SARS-CoV-2 virion is approximately 50 to 200 nanometres in diameter. Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. In some embodiments, the nucleic acid sequence of the severe acute respiratory syndrome coronavirus 2 isolate (SARS CoV2) Wuhan-Hu-1, complete genome is denoted by NCBI Reference Sequence: NC_045512.2. In yet some further embodiments, the SARS CoV2 nucleic acid sequence is as denoted by SEQ ID NO: 19. In the specific case of the SARS CoV2, a defined receptor-binding domain on S mediates the attachment of the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). In yet some further embodiments, the spike protein of said SARS CoV2, comprises the amino acid sequence as denoted by YP_009724390.1, and disclosed herein by SEQ ID NO: 20.

In some specific embodiments, a Coronavirus applicable in the present invention may be

Middle East Respiratory Syndrome coronavirus (MERS CoV).

The term MERS CoV as known in the art refers to a lineage C beta coronavirus (+RNA 30kb) whose primary natural reservoir resides in bats that infect domesticated camels as opportunistic hosts, which go on to infect humans. MERS-CoV genomes are phylogenetically classified into two clades, clade A and B. The earliest cases of MERS were of clade A clusters (EMC/2012 and Jordan-N3/2012), and new cases are genetically distinct (clade B). MERS-CoV is distinct from SARS-CoV and distinct from the common-cold coronavirus and known endemic human betacoronaviruses HCoV-OC43 and HCoV-HKUl. Until 23 May 2013, MERS-CoV had frequently been referred to as a SARS-like virus, or simply the novel coronavirus, and early it was referred to colloquially as the "Saudi SARS". Over 1,600 cases of MERS have been reported by 2015 and the case fatality rate is >30%. 182 genomes have been sequenced by 2015 (94 from humans and 88 from dromedary camels). All sequences are >99% similar. The genomes can be divided into two clades - A and B - with the majority of cases being cased by clade B. Human and camel strains are intermixed suggesting multiple transmission events.

Like other coronaviruses, the MERS-CoV virion utilizes a large surface spike (S) glycoprotein for interaction with and entry into the target cell. The S glycoprotein consists of a globular SI domain at the N-terminal region, followed by membrane -proximal S2 domain, a transmembrane domain and an intracellular domain.

Determinants of cellular tropism and interaction with the target cell are within the S 1 domain, while mediators of membrane fusion have been identified within the S2 domain. Through co- purification with the MERS-CoV SI domain, dipeptidyl peptidase 4 (DPP4) was identified as cellular receptor for MERS-CoV. DDP4 is expressed on the surface of several cell types, including those found in human airways, and possesses ectopeptidase activity, although this enzymatic function does not appear to be essential for viral entry. In yet some other alternative embodiments, the Coronavirus may be Severe Acute Respiratory Syndrome coronavirus (SARS CoV).

Specifically SARS-CoV has been associated with a viral disorder characterized by high fever, dry cough, shortness of breath (dyspnea) or breathing difficulties, and atypical pneumonia. The complete SARS-CoV has been analyzed and published by Marra et al. 2003. Since then a large number of SARS strains have been isolated and characterized, and are accessible via the Centers for Disease Control and Prevention (CDC), or the National Center for Biotechnology Information, e.g. the sequence of the SARS-CoV Urbani strain is under Acc. Num. AY278741.

More specially, the SARS coronavirus (SARS-CoV) is a lineage B beta coronavirus that causes severe acute respiratory syndrome (SARS). SARS-CoV is a positive and single stranded RNA virus belonging to a family of enveloped coronaviruses. Its genome is about 29.7 kb. The SARS virus has 13 known genes and 14 known proteins. SARS is similar to other coronaviruses in that its genome expression starts with translation of two large ORFs, la and lb, both of which are polyproteins. The functions of several of these proteins are known, ORFs la and lb encode the replicase and there are four major structural proteins: nucleocapsid, spike, membrane and envelope. It also encodes for eight unique proteins, known as the accessory proteins, all with no known homologues or function.

In yet some further embodiments, the devices, systems, and methods of the invention may be applicable for detecting bacterial pathogens in a sample.

The term "bacteria" (in singular a "bacterium") in this context refers to any type of a single celled microbe. Herein the terms "bacterium" and "microbe" are interchangeable. This term encompasses herein bacteria belonging to general classes according to their basic shapes, namely spherical (cocci), rod (bacilli), spiral (spirilla), comma (vibrios) or corkscrew (spirochaetes), as well as bacteria that exist as single cells, in pairs, chains or clusters.

It should be noted that the term "bacteria" as used herein refers to any of the prokaryotic microorganisms that exist as a single cell or in a cluster or aggregate of single cells. In more specific embodiments, the term "bacteria" specifically refers to Gram positive, Gram negative or Acid-fast organisms. The Gram-positive bacteria can be recognized as retaining the crystal violet stain used in the Gram staining method of bacterial differentiation, and therefore appear to be purple-colored under a microscope. The Gramnegative bacteria do not retain the crystal violet, making positive identification possible. In other words, the term 'bacteria' applies herein to bacteria with a thicker peptidoglycan layer in the cell wall outside the cell membrane (Gram-positive), and to bacteria with a thin peptidoglycan layer of their cell wall that is sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane (Gram-negative). This term further applies to some bacteria, such as Deinococcus, which stain Gram-positive due to the presence of a thick peptidoglycan layer, but also possess an outer cell membrane, and thus suggested as intermediates in the transition between monoderm (Gram-positive) and diderm (Gram-negative) bacteria. Acid fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids that resist staining by conventional methods such as a Gram stain.

Of particular interest, a pathogen to be detected by the devices, systems, and methods of the invention, may be any bacteria involved in nosocomial infections or any mixture of such bacteria. The term "Nosocomial Infections " refers to Hospital-acquired infections, namely, an infection whose development is favored by a hospital environment, such as surfaces and/or medical personnel, and is acquired by a patient during hospitalization. Nosocomial infections are infections that are potentially caused by organisms resistant to antibiotics. Nosocomial infections have an impact on morbidity and mortality, and pose a significant economic burden. In view of the rising levels of antibiotic resistance and the increasing severity of illness of hospital in-patients, this problem needs an urgent solution. Common nosocomial organisms include Clostridium difficile, methicillin-resistant Staphylococcus aureus, coagulase-negative Staphylococci, vancomycin-resistant Enteroccocci, resistant Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter and Stenotrophomonas maltophilia.

The nosocomial-infection pathogens could be subdivided into Gram-positive bacteria Staphylococcus aureus, Coagulase-negative staphylococci'), Gram-positive cocci (Enterococcus faecalis and Enterococcus faecium), Gram-negative rod-shaped organisms (Klebsiella pneumonia, Klebsiella oxytoca, Escherichia coli, Proteus aeruginosa, Serratia spp.), Gram-negative bacilli (Enterobacter aerogenes, Enterobacter cloacae), aerobic Gram-negative coccobacilli (Acinetobacter baumanii, Stenotrophomonas maltophilia) and Gram-negative aerobic bacillus (Stenotrophomonas maltophilia, previously known as Pseudomonas maltophilia). Among many others Pseudomonas aeruginosa is an extremely important nosocomial Gram-negative aerobic rod pathogen. In particular and non-limiting embodiments, such microbe of interest may be an antibiotic-resistant bacteria.

Of particular interest are “ESKAPE” pathogens. As indicated herein, these pathogens include but are not limited to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter.

Thus, in some embodiments of the invention relate to bacteria of any strain of at least one of E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Clostidium difficile, Enterococcus faecium, Klebsiella pneumonia, Acinetobacter baumanni and Enterobacter species (specifically, ESKAPE bacteria).

In further embodiments the pathogen according to the present disclosure may be a bacterial cell of at least one of E. coli, Pseudomonas spp, specifically, Pseudomonas aeruginosa, Staphylococcus spp, specifically, Staphylococcus aureus, Streptococcus spp, specifically, Streptococcus pyogenes, Salmonella spp, Shigella spp, Clostidium spp, specifically, Clostidium difficile, Enterococcus spp, specifically, Enterococcus faecium, Klebsiella spp, specifically, Klebsiella pneumonia, Acinetobacter spp, specifically, Acinetobacter baumanni, Yersinia spp, specifically, Yersinia pestis and Enterobacter species or any mutant, variant isolate or any combination thereof.

A lower eukaryotic organism applicable in the present invention as pathogens to be detected by the devices, systems, kits and methods provided by the invention, includes a yeast or fungus such as but not limited to Pneumocystis carinii, Candida albicans, Aspergillus, Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Trichophyton and Microsporum, are also encompassed by the invention.

A complex eukaryotic organism includes worms, insects, arachnids, nematodes, aemobe, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Trypanosoma brucei gambiense, Trypanosoma cruzi, Balantidium coli, Toxoplasma gondii, Cryptosporidium or Leishmania.

Still further, in certain embodiments the devices, systems, kits and methods of the invention may be suitable for detecting fungal pathogens. The term "fungi" (or a “fungus”), as used herein, refers to a division of eukaryotic organisms that grow in irregular masses, without roots, stems, or leaves, and are devoid of chlorophyll or other pigments capable of photosynthesis. Each organism (thallus) is unicellular to filamentous, and possess branched somatic structures (hyphae) surrounded by cell walls containing glucan or chitin or both, and containing true nuclei. It should be noted that "fungi" includes for example, fungi that cause diseases such as ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidio-idoiny cosis, and candidiasis.

As noted above, the present invention also provides for devices, systems, kits and methods that may be suitable for detecting a parasitic pathogen. More specifically, “parasitic protozoan”, which refers to organisms formerly classified in the Kingdom “protozoa”. They include organisms classified in Amoebozoa, Excavata and Chromalveolata. Examples include Entamoeba histolytica, Plasmodium (some of which cause malaria), and Giardia lamblia. The term parasite includes, but not limited to, infections caused by somatic tapeworms, blood flukes, tissue roundworms, ameba, and Plasmodium, Trypanosoma, Leishmania, and Toxoplasma species.

As used herein, the term “nematode” refers to roundworms. Roundworms have tubular digestive systems with openings at both ends. Some examples of nematodes include, but are not limited to, basal order Monhysterida, the classes Dorylaimea, Enoplea and Secernentea and the “Chromadorea” assemblage.

As shown by the following Examples, the methods provided by the invention are particularly applicable for detecting a viral pathogen, for example the SARS CoV-2 in a patient, thereby demonstrating the feasibility of using the methods of the invention for the diagnosis and monitoring of an infectious disease caused by at least one of the pathogen/s in a subject.

In more specific and non-limiting embodiments, the methods of the invention may be particularly applicable for detecting viral pathogens that may affect airways. In some particular embodiments, the methods of the invention may be applicable for the diagnosis of COVID-19 in a mammalian subject.

Subjects, as used by the invention, encompass any multicellular organism, for example, any living multi-cellular vertebrate organisms, a category that includes both human and non-human animals, such as non-human mammals (such as mice, rats, rabbits, sheep, horses, cows, and non-human primates).

More specifically, SARS-CoV2 has been associated with a viral disorder named COVID- 19 which is characterized by numerous symptoms, while the most common symptoms are fever and a dry cough. The third most common symptom is fatigue. Almost 40% of cases suffered from it. ‘Sputum production’ (a thick mucus which is coughed up from the lungs) was experienced by every third person. Sputum is not saliva. It is thick mucus which is coughed up from the lungs. About 18.6% experienced shortness of breath (‘dyspnoea’). Many of the most common symptoms are shared with those of the common flu or cold while SARS-CoV2 infection rarely causes a runny nose. According to the WHO, people infected with SARS-CoV2 generally develop signs and symptoms, including mild respiratory symptoms and fever, on an average of 5-6 days after infection. While the mean incubation period is 5 to 6 days, the WHO adds that the incubation period can vary in a wide range of between 1 to 14 days.

Symptoms were categorized as mild, severe, or critical and the research article describes these as follows:

Critical cases: Critical cases include patients who suffered respiratory failure, septic shock, and/or multiple organ dysfunction or failure, that may lead to death.

Severe cases: This includes patients who suffered from shortness of breath, respiratory frequency > 30/minute, blood oxygen saturation <93%, PaCh/FiCh ratio <300,30 and/or lung infiltrates >50% within 24-48 hours.

Mild cases: The majority (81%) of these coronavirus disease cases were mild cases. Mild cases include all patients without pneumonia or cases of mild pneumonia.

In some embodiments, the methods of the invention may be performed using any means and any suitable devices or systems. In some particular embodiments, the methods of the invention may be performed using any of the devices of the invention as defined herein before, or any of the systems of the invention as defined by other aspects of the invention herein before.

As shown by the Examples, the invention provides pathogen detection devices, assays and kits providing high sensitivity and specificity. The term "sensitivity" is used herein with respect to the ability of the methods of the invention to detect a pathogen, specifically, amplification products that use as a template nucleic acid sequences of a target pathogen, even when said nucleic acid sequence template is present in a low concentration in a sample. In some particular embodiments, the sensitivity of the methods of the invention may detect concentration that may be less than viral load equivalent to Ct qRTPCR cycle of 28 and under.

The term "specificity" is used herein with respect to the ability of the devices, and methods of the invention to detect a pathogen, specifically, amplification products specific for such pathogen, by preferentially using as a template nucleic acid sequences specific for said target pathogen in a sample. The specific primers used by the reaction mixtures used by the invention bind the intended nucleic acid sequence of the target pathogen for use as a template to produce a specific amplification product, at least 1.5 fold more than any other nucleic acid sequence in said sample, at least 2.0 fold, at least 2.5 fold, at least 5.0 fold, at least 10.0 fold, at least 20.0 fold, at least 50.0 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1000 fold, at least 5000 fold, at least 10000 fold, at least 50,000 fold, or at least 100,000 fold.

It should be understood that in some embodiments, all the components of the modules, devices of the invention may be provided either separately, or in any mixtures thereof. For example, in cases where the kit comprises the cheotropic agent for use with the at least one protease, both components may be provided either separately, or together as a mixture dried (e.g., lyophilized) forms thereof. Another example is the components of the reaction mixture, that may be provided either with or without the at least one cheotropic agents, that may be provided either separately, or as a mixture or any other combination. More specifically, in some embodiments, each and every component of the reaction mixture, with or without the cheotropic agents may be provided separately. In yet some alternative embodiments, all components or any combinations thereof, may be provided together as a mixture in a dried (e.g., lyophilized) forms thereof.

As indicated above, all components of the devices of the invention can be provided separately. In some embodiments, all components and reagents of the module and devices discussed herein, specifically those required for performing the methods of the present disclosure may be provided in a kit. In some embodiments, the different components of the devises of the invention may be provided in separate compartments, or any other separating means. However, it must be understood that in some alternative embodiments, the amplification zones of the sample testing module of the invention, or any device, or systems of the invention, may be in the form of an array. Thus, in some embodiments, the different components of the kits of the invention may be provided separately, in an array. The term "array" as used by the invention refers to an "addressed" spatial arrangement of the plurality of reaction mixtures. Each "address" of the array is a predetermined specific spatial region containing said reaction mixture. An array that comprise such plurality of amplification zones may also be any solid support holding in distinct regions (dots, lines, columns) different reaction mixtures. The array preferably includes built-in appropriate controls, for example, regions without the sample, regions without various reagents of the reaction mixture, for example, polymerase, dNTPs, the specific pH-sensitive dye, intercalating compounds, the chaotropic agent, or any combinations thereof, or even with buffers alone.

As indicated above, the reaction mixture provided by the reaction zones of the invention is specific for providing an amplification product of a particular nucleic acid sequence of at least one pathogen. However, in some embodiments, control reactions mixtures may be also included by the amplification zones of the invention. More specifically, in some embodiments, at least one of the reaction mixtures provided by the kit of the invention may comprise at least one set of primers specific for at least one control nucleic acid sequence. In yet some further embodiments, reaction mixture specific for at least one control nucleic acid sequence, may be provided comprised within at least one second reaction chamber. Such control reaction mixture may comprise at least one set of primers specific for at least one control nucleic acid sequence.

In some embodiments, the LAMP reaction mixture further comprises at least one pH sensitive indicator dye. It should be noted that such dye provides a detectable signal upon production of at least one amplification product by the amplification reaction.

In some embodiments, the pH sensitive indicator dye is a colored dye detectable in visible light.

In yet some alternative embodiments, the pH sensitive indicator dye may be a fluorescent indicator dye. In some alternative embodiments, the kit of the invention provides reaction mixture that supports production of amplification product that comprises a detectable agent. Such detectable compound may be added directly or indirectly to one of the components of the produced amplification product. In some embodiments, such detectable agent may be an agent that is intercalated into the amplification product. In some embodiments, the LAMP reaction mixture further comprises at least one fluorescent compound capable of intercalating into a double strand DNA, thereby providing a detectable signal upon production of at least one amplification product. In some embodiments, such fluorescent compound that intercalates into the nucleic acid amplification product may be SYBER green.

In yet some alternative embodiments, the reaction mixture provided by the kits of the invention may comprise as a compound that intercalates into the amplification product, an analogue of thymidine. In more specific embodiments, such thymidine analog may be 5-Ethynyl-2'-deoxyuridine (EdU). More specifically, EdU is a thymidine analog that can be incorporated into the amplified product. In yet some further embodiments, the EdU may be detected through a click chemistry reaction.

In yet some further alternative embodiments, the kit of the invention may be adapted for detecting the formation of amplification products by measuring pH of the reaction. Thus, in accordance with some embodiments, the kit of the invention may comprise at least one means for detecting or measuring pH, for example, a pH meter or any other suitable pH sensor and recorder, other image analysis or any computerized application thereof.

In yet some further embodiments, the kit of the invention may be adapted for detecting the formation of amplification products by measuring a change in electric charge caused by release of hydrogen and production of a hydrogen potential, or production of pyrophosphate. The kit of the invention may further comprise in accordance with some embodiments, at least one membrane that enables accumulation of protons. In some other embodiments, the kit of the invention may be adapted for detecting the formation of amplification products by measuring or detecting a change in electrical resistance with respect to each amplification zone. For example, such changes in electrical resistance may be measured via sensing electrodes provided in the vicinity of the respective amplification zones; for example, two electrodes can be placed outside each respective amplification zone, facing one another across the respective amplification zone.

Still further, in some embodiments, the device of the invention is adapted for detection and monitoring of at last one pathogen in at least one sample.

In yet some further embodiments, such sample may be any sample, for example, at least one of a biological sample or an environmental sample. Specifically, any of the samples disclosed by the invention as detailed herein before in connection with other aspects.

In some embodiments, the device of the invention is applicable for detecting any pathogen. It should be understood that any pathogen disclosed by the invention is also applicable for any of the kits of the invention. According to some specific embodiments, such pathogen may be a viral pathogen.

In yet some further embodiments, the device and methods of the invention may be applicable for detecting a viral pathogen in a sample, such viral pathogen in accordance with some embodiments may be at least one corona virus (CoV).

Specific embodiments of the invention relate to devices and methods adapted for detecting a CoV that is SARS CoV-2.

The devices and methods of the invention are applicable for detecting pathogens in a sample. In some embodiments, specifically where the sample is obtained from a subject, particularly, a mammalian subject, the devices and methods may be useful for the diagnosis and/ or monitoring of at least one infectious disease caused by at least one pathogen in a subject. In some embodiments, such subject may be a mammalian subject.

In some embodiments, the devices and methods of the invention may be adapted for diagnosis and/ or monitoring of at least one infectious disease in a subject, performed using any of the methods of the invention, specifically as described in connection with other aspects of the invention. In some particular and non-limiting embodiments, the devices and methods of the invention may be applicable and therefore adapted for the diagnosis and/or monitoring of COVID-19 in a mammalian subject.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of’ “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Throughout this specification and the Examples and claims which follow, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open- ended, i.e., to mean including but not limited to. Specifically, it should understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures. More specifically, the terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

EXAMPLES

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.

Experimental procedures

Fabrication of superh vdrophobic surface

Polydimethylsiloxane (PDMS) (DOWSIL SYLGARD 184 Silicone Elastomer), was spin-coated (LAURELL WS-650SZ-6NPP/LITE) in 1:10 ratio, on a microfabricated silicon mold to obtain micro-structured surfaces. The product was let cure for 5hr at 60°C. The surfaces were composed of square micropillars (5pm X 5pm) organized in a square lattice with pillar spacing of 5, 10 and 15 pm. A nano structure Was next added to the pillars using chemical deposition of a 50mg of polyvinylchloride (PVC) (Sigma-Aldrich, polyvinyl chloride, high molecular weight, product number 81387), dissolved in a ethanol AR (Sigma-Aldrich St. Louis, MO), and tetrahydrofuran (THF) (Sigma-Aldrich St. Louis, MO) 1:1, 20ml mixture. In the deposition process, the solution was applied to the surfaces twice. After each time, the solvents were evaporated before brushing the deposited compound into the microstructure using a regular painting brush. The resulting surfaces were -125 pm thick and were cut to strips of 0.4cm X 1cm.

Samples collection:

Swabs from both throat and nose are previously collected to one tube by healthcare providers and sent to the Virology laboratory at the Rambam Health Care Campus, Haifa, Israel. The swabs were stored in 1-2 ml of Universal Transport Medium (UTM). Saliva samples are self-collected directly into sterile cups and kept at 4 °C until tested.

Quantitative reverse transcription PCR (RT-qPCR):

Viral RNA is extracted by either of three automated nucleic acid extraction systems: (1) easyMAG ® / EMAG (Biomeriuex); (2) magLEAD® 5bL (Precision System Science); (3) MagEx (STARlet). The following protocol is used: (1) 2 ml lysis buffer, 0.5 ml sample and 50 pl elution buffer; (2) 270 pl lysis buffer, 130 pl sample and 50 pl elution buffer; (3) 300 pl lysis buffer, 400 pl sample and 50 pl elution buffer, respectively. Following viral RNA extraction, the sample is placed on the sample preparation zone of the device and RT-qPCR is performed on the amplification rection zone of the device of the invention, using reagents of either of two commercial kits: (1) Allplex™ 2019-nCoV (Seegene); (2) Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV (BGI), according to manufacturer’s instructions. Additional RT-qPCR reaction mix was created manually, using custom made primers (Primers table) as follows: The probe IC was synthesized with a 5' FAM/CY5 Fluorophore moiety and 3' ZEN/IBFQ quencher. Each of the manual reactions was assembled in a total volume of 25 pl, in each o the reaction zones of the disclosed device. Specifically, 12.5pl 2X Ag-Path One-step mix (Ambion), 1 pl primers, Ipl of Reverse Transcriptase enzyme, 5 pl of the sample’s RNA extracted previously and H2O to a final volume of 25 pl. GeneN-A primers published in Zhang et al., [ Zhang, Y. et al. Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv, 2020.2002.2026.20028373, doi: 10.1101/2020.02.26.20028373 (2020)].

RNase P POP7 primers published in Curtis et al., [Curtis, K. A. et al. A multiplexed RT- LAMP assay for detection of group M HIV-1 in plasma or whole blood. Journal of Virological Methods 255, 91-97 (2018)].

Table 1: Primer table (sequences of the specified primers used herein are denoted by SEQ ID NO: 1 to 18).

Colorimetric RT-LAMP Reaction:

5 j l of UTM samples are diluted in 40 pl of DNase RNase free water (Biological Industries, 01-869-1B) and 2 pl of Proteinase K (1.22 mg/ml final concentration) (Seegene, 744300.4.UC384). Samples are placed in the preparation zone of the disclosed device and are incubated at room temperature for 15 minutes. Inactivation of proteinase K is obtained by incubating the reaction tubes at 95 °C for 5 minutes, according to step (b) of the disclosed method. Next, following transfer of aliquots of the prepared sample to the amplification reaction zone of the device, colorimetric RT-LAMP reaction is performed in a total volume of 20 pl per reaction using 10 pl WarmStart® Colorimetric LAMP 2X Master Mix (New England BioLabs Inc., Ml 800), 2 pl primers mix (see Primers Table), 1 pl Guanidine hydrochloride (Sigma, G4505), to a final concentration of 40 mM, and 7 pl of the inactivated sample. The reaction is then incubated for 30-40 minutes at 65 °C. Test samples were interpreted without prior knowledge of the reference standard results.

Statistical analysis:

TPR (true positive rate), TNR (true negative rate), FPR (false positive rate), FNR (false negative rate) were calculated according to the following equations: TPR= TP/(TP+FN). TNR=TN/(FP+TN). FNR=FN/(TP+FN). FPR=FP/(FP+TN). TP: total number of true positives. TN: total number of true negatives. TN: total number of true negatives. FN: total number of false negatives.

Ethical approval:

This study was granted exemption from IRB approval of the Rambam Health Care Campus for use of de-identified COVID-19 tests performed for the purpose of the standard testing, and for 4 volunteers.

EXAMPLE 1

Optimization of RT-LAMP for the detection of SARS-CoV-2 RNA directly from crude human patient swab samples, without RNA purification steps

The inventors first performed RT-LAMP on RNA purified from COVID- 19-positive and -negative swabs. The primers used in the present examples, were previously designed and validated by Zhang et al. [4] (see primers in Table 1). Samples were studied from positive and negative patients for the SARS-CoV-2. The samples were confirmed by approved RNA purification and quantification at the Rambam Health Care Campus (RHCC) hospital.

LAMP results can be visualized by color change. RT-LAMP was first applied on purified RNA from COVID-19 positive and negative swabs. As in Zhang et. al., [4], the RT- LAMP results agreed with the standard RT-qPCR results. To simplify the detection method, the RT-LAMP reaction is tested on crude throat and nose swabs from patients. These swabs are kept in universal transfer media (UTM). For crude samples, an optional inactivation step (before step (a) of the disclosed method)is added by heating the UTM to 95°C for 5 minutes. This RT-LAMP protocol is evaluated on a cohort of 99 patients that were tested at the hospital. This pool included 27 positive samples with a wide range of viral load and 72 negative samples. Samples previously evaluated by the standard RT- qPCR test. Since crude samples may contain enzymatic inhibitors that might affect the efficiency of viral RNA detection, the addition of proteinase K and guanidine hydrochloride to the process is tested during the sample preparation step performed in the preparation zone of the disclosed device. Proteinase K is provided in the preparation zone of the device and thus is contacted with the crude UTM sample taken from the original tube. Guanidine hydrochloride is provided in the reaction zone for the RT-LAMP reaction step.

EXAMPLE 2

Validation Analysis an additional cohort of patients suspected of SARS-CoV-2

This adjusted protocol is further validated on an additional cohort of 83 patients suspected of SARS-CoV-2. These patients are tested at RHCC by the standard RT-qPCR, 31 were negative and 52 were positive with a wide range of Ct values (14-35). It is of interest to find the optimal incubation time in the amplification zone of the disclosed device to yield the best rate of true positives without increasing the rate of false negatives. The samples are applied on the device of the present disclosure and RT-LAMP reaction is performed for up to 40 minutes and evaluated the colorimetric results at time -points 30, 35 and 40 minutes.