DIAGNOSTIC SIGNAL

TECHNICAL FIELD

[0001] The present disclosure generally relates to the field of diagnostics, and, in particular, to detecting infectious pathogens in biological samples. More particularly, the present disclosure provides methods, assays, kits and devices for detecting the presence or absence of an inactivated, less infective form of an infectious pathogen in a biological sample.

BACKGROUND

[0002] There is a significant need to test asymptomatic or symptomatic individuals for infectious pathogens, such as viruses that may cause viral infections. Precautions are required during collecting and handling of these potentially infectious biological samples. This is a significant hazard for the medical and lab personnel that are exposed to large numbers of such samples.

[0003] In addition, there is a need to inactivate the infectivity of potentially infective biological samples without affecting the ability to detect the pathogens that may be present in the sample. Accordingly, there is a need for methods, devices, assays and kits that can reduce or eliminate infectivity of a biological sample while preserving the samples ability to be analyzed for the presence of various pathogens. Meeting this need will allow health care professionals to analyze samples to diagnose diseases caused by infectious pathogens without the risk of contracting the pathogens themselves.

[0004] Treatment with heat or radioactivity can be used for the general inactivation of infectious samples; however, such methods may cause reduction of the detectable signal through damage of the biological materials. These approaches are undesirable because they may lead to an increased proportion of false negative test results. These approaches are also impractical for large scale, safe adoption, without the use of highly specialized equipment.

[0005] Detection of microbial pathogens in biological samples is of particular value in clinical medicine, as treatment may vary considerably depending upon the causative organism. Thus, the accurate and rapid identification of pathogens in biological samples of patients suspected of having an infectious disease can be critical to provide prompt and appropriate treatment to patients. Rapid identification of disease-causing organisms in biological samples is important even for non-life threatening infections.

[0006] Rapid methods of diagnosing microbial infections have been developed to provide timely results for guiding clinical therapy. Some of the most effective of these rapid methods have been immunologically based. Monoclonal and polyclonal antibodies to microbe-specific antigens have been developed and used in immunoassays to identify specific microbes in biological samples.

[0007] Accordingly, there remains a need for reducing and/or eliminating the infectivity of a biological sample without interfering with the ability to rapidly detect pathogens present in the sample for diagnostic purposes.

BRIEF SUMMARY

[0008] The present disclosure provides devices, methods, assays and diagnostic kits for detecting the presence or absence of an inactivated form of an infectious pathogen in a biological sample.

[0009] In one aspect, the present disclosure provides a method for detecting presence or absence of an inactivated form of an infectious pathogen in a biological sample, comprising combining a biological sample suspected of comprising an infectious pathogen with a processing reagent comprised of a photoreactive compound. In some embodiments, the present disclosure provides exposing the biological sample with the processing reagent to an ultraviolet photon from ultraviolet A, B or C (UVA, UVB, or UVC) light to create a test sample. In some embodiments, if the infectious pathogen is present in the biological sample, the test sample comprises pathogen with a reduced infectivity, and the pathogen with reduced infectivity is detectable in a detection assay for the infectious pathogen.

[0010] In another aspect, the present disclosure provides a method for reducing infectivity of a biological sample suspected of comprising an infectious pathogen, comprising: providing a processing reagent and combining or instructing to combine a biological sample suspected of comprising an infectious pathogen and the processing reagent. In some embodiments, the biological sample comprising the processing agent is illuminated to produce a test sample with substantially all pathogen in a non-infectious form. In other embodiments, the test sample is optionally tested in an assay to determine presence or absence of pathogen, wherein the assay measures pathogen in the non-infectious form with a sensitivity and/or specificity essentially the same as the infectious pathogen. In other embodiments, the presence or absence of pathogen is determined based on the results of said testing.

[0011] In another aspect, the present disclosure provides a method for detecting a non-infectious form of an infectious pathogen in a sample. In some embodiments, a diagnostic device for detection of an infectious pathogen is provided. In some embodiments, a processing reagent is provided that renders substantially all infectious pathogen in a sample to a non-infectious form. In other embodiments, detection of the non-infectious form of the pathogen on the diagnostic device is provided. [0012] In another aspect, the present technology provides an assay, comprising a container with a processing reagent comprised of a photoreactive compound capable of rendering an infectious pathogen non-infectious. In some embodiments, the assay further comprises a tool for collection of a biological sample. In some embodiments, the assay further comprises a device for detection of an infectious pathogen. In other embodiments, the assay further includes an illumination source. In additional embodiments, the assay also includes instructions to use the tool to collect a sample suspected of comprising an infectious pathogen, to place the sample or the tool with the sample into the container, to expose the container to the illumination source, and to remove the sample from the container and place it on the device. In some embodiments, exposure of the container containing the biological sample and the photoreactive compound to the illumination source renders substantially all of the infectious pathogens non-infectious.

[0013] It is envisioned that the photochemical technology (PCT) virus inactivation techniques, of the present technology are compatible with various configurations of sampling devices/tools, reagent reservoirs, extraction and incubation chambers, sample application means, and downstream analytical devices (such as lateral flow immunoassay devices or test strips). For example, a processing reagent comprising a photoreactive compound may be present in or on any of the tools, compartments, reservoirs, or chambers for sample collection, processing or analysis. Also, the various tools, compartments, reservoirs, or chambers for sample collection, processing or analysis may be manufactured from materials that allow transmission of ultraviolet A, B and/or C light, such as plastics that allow transmission of UV light. In some embodiments, the various tools, compartments, reservoirs, or chambers for sample collection, processing or analysis that are compatible with the presently described PCT technology are designed for a single use. In other embodiments, the various tools, compartments, reservoirs, or chambers for sample collection, processing or analysis that are compatible with the presently described PCT technology are designed for more than one use.

[0014] In another aspect, the present technology provides a method for detecting presence or absence of an inactivated form of an infectious pathogen in a biological sample by exposing the biological sample to ultraviolet B or C light to create a test sample. In some embodiments, a processing agent, such as a photoreactive compound, is added to the sample before exposure to the ultraviolet light. In other embodiments, no processing agent is added to the sample prior to exposure of the sample to UVB or UVC light. In some embodiments the UVB or UVC light comprises a wavelength of about 250 nm to about 325 nm or from about 275 nm to about 300 nm. In some embodiments, if the infectious pathogen is present in the biological sample, the test sample comprises pathogen with a reduced infectivity. In some embodiments, the pathogen with the reduced infectivity is detectable in a detection assay for the infectious pathogen.

[0015] In some aspects, the present technology provides a processing reagent that is a photoreactive compound. In some embodiments, the photoreactive compound is a DNA or RNA intercalating compound. In some embodiments, the photoreactive compound is a furanocoumarin compound, such as a linear furanocoumarin compound. In some embodiments, the linear furanocoumarin compound is psoralen, angelicin, 5-geranoxypsoralen, xanthotoxin, bergapten, nodakenetin, 8-methoxy psoralen, or amotosalen.

[0016] In some aspects, method of the present technology include exposing a biological sample to ultraviolet light. In some embodiments, the ultraviolet light is ultraviolet A (UVA) light, ultraviolet B (UVB) light, or ultraviolet C (UVC) light. In some embodiments, the ultraviolet light is emitted from a light emitting diode (LED). In some embodiments, the ultraviolet light source is a reusable light source. In other embodiments, the ultraviolet light source is a disposable light source. In some embodiments, the ultraviolet light is ultraviolet light with a wavelength in the range of about 250 nm to about 430 nm. In other embodiments, the ultraviolet light is ultraviolet light with a wavelength of about 356 nm. In other embodiments, the ultraviolet light is ultraviolet light with a wavelength in the range of about 280 nm to about 430 nm or from about 280 nm to about 400 nm.

[0017] In some aspects, the biological sample is exposed to ultraviolet light prior to placing the test sample on the detection assay. In some embodiments, the biological sample is placed in a vial containing the processing reagent and the vial containing the biological sample and the processing reagent is exposed to an ultraviolet light source. In some embodiments, the ultraviolet light source is disposable. In other embodiments, the vial is plastic, such as a polypropylene or polyethylene plastic. In some embodiments, the biological sample is exposed to ultraviolet light by an instrument that is part of the detection assay.

[0018] In some aspects, the methods provided herein include a detection assay that is an immunodetection assay. In some embodiments, the immunodetection assay is an instrumentread immunodetection assay. In some embodiments, the detection assay is a nucleic acid detection assay. In some embodiments, the provided nucleic acid detection assays may include isothermal amplification of nucleic acid or thermal cycling amplification of nucleic acid.

[0019] In another aspect, the present disclosure provides detection of reduced infectivity pathogens in biological samples, wherein the pathogen is a virus, a bacterium, or a protozoa. In some embodiments, the pathogen is a DNA virus, RNA virus or RNA enveloped virus. In some embodiments, the pathogen is a DNA virus, RNA virus or RNA enveloped virus. In some embodiments, the pathogen is a virus selected from influenza A, influenza B, and SARS CoV-2. In some embodiments, the biological sample is whole blood, plasma, serum, saliva, or mucous. In some embodiments, the biological sample is present on a swab, and the swab is, optionally, combined with a processing reagent, for example in a vial, and then exposed ultraviolet light. In some embodiments, the sample is processed with a detergent containing solution that assists or accelerates pathogen inactivation.

[0020] The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.

BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 provides a schematic illustration of a method wherein a biological sample containing an infectious pathogen is processed in such a manner to remove infectivity of the sample without interfering with subsequent diagnostic testing by lateral flow immunoassay.

[0022] FIG. 2 provides a graph comparing detection of SARS CoV-2 antigen from a photochemical technology (PCT) inactivated SARS CoV-2 sample and a positive control, untreated/infectious SARS CoV-2 sample. The graph shows amount of virus (log pfu) versus signal/ cut-off (S/CO) as detected by SOFIA®2 for the PCT inactivated and untreated/infectious samples.

[0023] These and other embodiments are further described in the detailed description that follows.

DETAILED DESCRIPTION

I. Definitions

[0024] Before the present methods, assays, kits and devices are described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. Several embodiments of the present disclosure are described in detail hereinafter. These embodiments may take many different forms and should not be construed as limited to those embodiments explicitly set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0025] All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety.

[0026] As used herein, the following terms are intended to have the following meanings: [0027] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of such proteins and reference to "the formulation" includes reference to one or more formulations and equivalents thereof known to those skilled in the art, and so forth.

[0028] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed by this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed by this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also within the scope of this disclosure.

[0029] “Protein,” “polypeptide,” “oligopeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids. In some embodiments, proteins and/or peptides may provide the basis for pathogen detection and diagnosis of pathogen associated diseases or disorders.

[0030] As pertains to the present disclosure, a “biological sample” may be a biological fluid and can be a solid, or semi-solid sample, including feces, biopsy specimens, skin, nails, and hair, or a liquid sample, such as urine, saliva, sputum, mucous, blood, blood components such as plasma or serum, amniotic fluid, semen, vaginal secretions, tears, spinal fluid, washings, and other bodily fluids. Biological samples also include extracts of such biological fluids in aqueous or nonaqueous matrices. Included among the sample are swab specimens from, e.g., the cervix, urethra, nostril, and throat. Any of such samples may be from a living, dead, or dying animal or a plant. Animals include mammals, such as humans.

[0031] “Detect” and “detection” have their standard meaning, and are intended to encompass detection, measurement and/or characterization of a selected protein or protein activity. For example, enzyme activity may be “detected” in the course of detecting, screening for, or characterizing inhibitors, activators, and modulators of the protein.

[0032] The term “reference level” refers to a detected level of a positive or negative control. For example, a reference level of a positive control can be a known amount of pathogenic antigen, obtained from a sample or culture of a known pathogen, a subject known to be infected with said pathogen, or can refer to a numerical value derived from known sources of pathogen-specific antigen.

II. Detection of Pathogens from Samples with Reduced Infectivity

[0033] The methods, provided herein provide an alternative to heat and radiation for reducing infectivity of biological samples. For example, in some embodiments, the present technology provides, as an alternative for reducing biological sample infectivity, the use of nucleic acid targeting with ultraviolet (UV) light-activated small molecules.

[0034] In some embodiments, the methods provided herein can be used without the destruction of the protein and nucleic acid components that provide the basis for subsequent diagnostic pathogen testing. For example, the present disclosure provides, in at least one embodiment, detection of inactivated pathogens, such as SARS COV-2 virus, at levels as low as 0.27 plaque forming units (pfu)/mL.

[0035] In some embodiments, the present technology provides modified extraction reagents that include a processing reagent. In some embodiments, the processing reagent incorporates a photoactive compound that can be used with ultraviolet A (UVA) or ultraviolet B (UVB) light to inactivate the infectivity of clinical or other biological samples before the application of the sample on a point of care (POC) detection assay, such as a lateral flow immune assay or cassette for pathogen detection.

[0036] In some embodiments, the processing reagents provided herein may include amotosalen, 8-methoxy psoralen, or other psoralens or photoactivatable, DNA binding compounds. In some embodiments, the processing reagents provided herein are compound stock solutions that are stable at room temperature (about 25 °C) in solution for more than about a year and could be lyophilized for an increased stability.

[0037] In other embodiments, the present technology includes a modified biological sample extraction composition including extraction components combined with processing reagents, such as photoactive reagents. Such modified extraction compositions can be used, in some embodiments, in a manner that is consistent with standard extraction procedures with the addition of a UV light illumination step, such as UVA, UVB or UVC illumination. In some embodiments, UV illumination of a biological sample is performed by exposing a sample in a disposable tube interfacing with a reusable UV source and holder, through plastic layer(s). Alternatively, such UV treatment could be incorporated in a customized sample taking device incorporating a UV source that is disposable. [0038] Other sources of illumination, such as ultraviolet B (UVB) or ultraviolet C (UVC) light are also provided herein with, or without the use of an additional processing agent, such as a photochemical agent. For example, the current disclosure also provides methods of reducing or eliminating infectivity of a biological sample, prior to diagnostic testing, by exposing the biological sample to UVC light in the presence or absence of the presently described processing reagents.

[0039] Methods, assays, devices and diagnostic kits for detecting the presence or absence of an infectious agent in a biological sample wherein the biological sample has reduced infectivity are provided. The methods described herein provide treatment of infectious samples in a manner that reduces infectivity of the samples but preserves the ability to detect the presence of the pathogen in the sample.

[0040] The extraction solution can be lyophilized, or dried down, to maintain the integrity of the components and the extraction capacity during the storage at 2-8 °C or room temperature (about 25 °C). The lyophilized cake, or dried powder, can be rehydrated before testing. Rehydration of the extraction reagent can be achieved with transfer medium, a buffer solution, or water.

[0041] In some embodiments, prior to using an assay or diagnostic device to detect the existence or absence of a pathogen in a biological sample, the biological sample is treated with the processing reagent, such as an intercalating agent. The processing reagents contemplated herein include, but are not limited to, DNA or RNA intercalating compounds such as furanocoumarin compounds, including linear furanocoumarin compound such as psoralen, angelicin, 5- geranoxypsoralen, xanthotoxin, bergapten, nodakenetin, 8-methoxy psoralen, or amotosalen.

[0042] The processing agent may either be in the form of a lyophilized or dried composition or in the form of liquid composition, comprising an intercalating reagent, for example, or a derivative thereof (such as a furanocoumarin compound). In some embodiments, the lyophilized or dried composition further comprise a salt, a sugar, a carrier protein (such as casein and/or methylated bovine serum albumin (BSA)), and/or detergents. In some embodiments, the lyophilized or dried composition even further comprises an antibody (polyclonal/monoclonal) specific to pathogen antigens coupled with microbeads (europium chelate-impregnated microbeads, or colored or magnetic microbeads) or gold sol, or carbon microparticles. In some embodiments, the salt is selected from sodium chloride, sodium phosphate, ammonium phosphate, magnesium sulfate, sodium acetate, sodium lactate, sodium succinate, sodium propionate, and potassium phosphate. In some embodiments, the sugar is selected from trehalose, sucrose, maltose, fructose, raffinose, lactose, and glucose. In some embodiments, the detergent is selected from TWEEN® 20, zwittergent, TRITON® X-100, and PLURONIC™ F-68. The processing reagent in the form of a liquid composition can be obtained by rehydrating the lyophilized or dried composition comprising a furanocoumarin compound or a derivative thereof as disclosed herein with a buffered solution (such as phosphate buffer, BIS-TRIS, MOPES, HEPES), water, or transfer medium (such as Arnie’s, Stuart, UTM).

[0043] The lyophilized or dried composition comprising a processing reagent or a derivative thereof can be prepared by lyophilizing a processing reagent in the form of an aqueous composition comprising, in addition to a furanocoumarin compound or a derivative thereof, a salt to maintain the pH, for example in the range of 6-8, a sugar, a carrier protein (such as casein and/or methylated BSA), and/or detergents. In some embodiments, the aqueous composition further comprises an antibody (polyclonal/monoclonal) specific to pathogen antigens coupled with microbeads (europium chelate-impregnated microbeads, or colored or magnetic microbeads) or gold sol, or carbon microparticles. In some embodiments, the salt is selected from sodium chloride, sodium phosphate, ammonium phosphate, magnesium sulfate, sodium acetate, sodium lactate, sodium succinate, sodium propionate, and potassium phosphate. In some embodiments, the sugar is selected from trehalose, sucrose, maltose, fructose, raffinose, lactose, and glucose. In some embodiments, the detergent is selected from TWEEN® 20, zwittergent, TRITON® X-100, and PLURONIC™ F-68.

[0044] In one embodiment, a method of using the processing reagent is provided. In the method, a specimen or biological sample from a patient suspected of a pathogen infection is provided. The patient specimens can be collected by, for example, a swab. The swab can be of any biological fluid, and examples include mouth, saliva, throat, lung, and nasal. The specimen swab can be tested directly by first rehydrating the lyophilized, or dried, processing reagent then adding the swab directly to the processing reagent. Alternatively, the specimen can be eluted from a swab into a transfer medium, which can then be used to rehydrate the processing reagent. [0045] The biological sample (swab or the transfer medium) can be incubated in the processing reagent, such as rehydrated processing reagent containing a furanocoumarin compound for a period of about 10 seconds to 20 minutes, 30 seconds to 15 minutes, 30 seconds to 10 minutes, 1-10 minutes, 1-8 minutes, 1-6 minutes, 2-10 minutes, 2-8 minutes, 2-6 minutes, or less than about 10 minutes, less than about 8 minutes, less than about 6 minutes, or for about 5 minutes or less.

[0046] The processing reagent incubated biological sample, may then be exposed to UV light, such as UVA, UVB and/or UVC light for a period of 30 seconds to 5 minutes. After incubation of the sample and exposure to UV light, the biological sample will be used as an antigen source for an immunoassay such as a pathogen specific lateral flow test strip, including, but not limited to SARS CoV-2 lateral flow strip tests.

[0047] In some embodiments, the enzymatic extraction method comprises mixing a biological sample containing or suspected of containing pathogen with the processing reagent in the form of a liquid composition as disclosed herein. In some embodiments, the processing reagent in the form of a liquid composition as disclosed herein is obtained by rehydrating the processing reagent in the form of a lyophilized or dried composition as disclosed herein with a buffered solution, water, or transfer medium. In some embodiments, the method comprises eluting the biological sample into a transfer medium followed by mixing the transfer medium with the processing reagent in the form of a lyophilized or dried composition as disclosed herein. In some embodiments, the method further comprises incubating the resulting mixture for a period of 1 to 10 minutes. At the completion of the extraction, the sample is exposed to ultraviolet light and then is applied to an immunoassay, such as a lateral flow based assay, for the pathogen signal detection.

[0048] Immunoassays for detection of infectious pathogen that involves pre-treating a biological sample containing or suspected of containing infectious pathogen with the processing agent as disclosed herein are contemplated, wherein the assay comprises a lateral flow device that allows for one-step pretreatment and detection of pathogenic organisms with enhanced specificity. Immunoassay devices are known in the art, and typically have at least a sample receiving zone, a labeling zone and a capture zone, and can be prepared according to the description in any of U.S. Pat. Nos. 5,415,994; 5,763,262 and 5,770,460, which are incorporated by reference in their entirety.

[0049] Molecular assays for detection of infectious pathogens are also contemplated, where the assay comprises a processing reagent for treating a biological sample containing or suspected of containing infectious pathogen and a contained with reagents for molecular amplification of a target sequence of the pathogen. In one embodiment, the reagents for amplification of the target sequence are for thermal amplification, and in another embodiment, the reagents for amplification are for isothermal amplification, such as helicase dependent amplification. The reagents for molecular amplification are well known to a skilled artisan and include a suitable enzyme for separating the strands of DNA, reverse and forward primers, and a labelled probe that binds an amplicon.

[0050] Accordingly, in one aspect of the disclosure, a device is provided for detecting the presence of pathogen in a sample, wherein the sample contains pathogenic molecules having no, or reduced, infectivity. Various embodiments of a device are contemplated, and exemplary embodiments are described herein for the purposes of illustration. A skilled artisan will appreciate, however, that the illustrative embodiments are non-limiting to the inventive concepts set forth herein.

[0051] In one embodiment, the lateral flow immunoassay comprises an immunoassay with label that can be read visually with the unaided eye, such as a colored bead or particle, wherein a collection of such beads or particles at the test line of the immunoassay can be viewed by eye. In another embodiment, the lateral flow immunoassay comprises an immunoassay with a label that is read by an instrument or by an eye with the aid of an instrument. For example, a fluorescent label in the immunoassay is detected using an instrument that can excite the label and the excited label can be read with the instrument, with the eye aided by instrument or with the eye. An exemplary instrument and lateral flow immunoassay is described in U.S. Patent No. 9,207,181, which is incorporated by reference herein.

[0052] Another embodiment of a device contemplated for use is described in U.S. Patent No. 5,415,994, which is incorporated by reference herein. In this embodiment, the device comprises a receiving chamber positioned, or capable of being positioned, for fluid contact with a lateral flow immunoassay device, and preferably positioned for fluid communication with a sample receiving zone or a labeling zone of the immunoassay test strip. The biological sample suspect of containing an infectious pathogen is received into the receiving chamber, such as by insertion of a swab containing the sample or by dispensing an aliquot of the sample into the receiving chamber. One or more processing reagents as disclosed herein can be additionally added to the receiving chamber or to the swab. In one embodiment, the biological sample comprising a processing reagent is exposed to ultraviolet light. In one embodiment, the receiving chamber is positioned over the sample receiving zone is dimensioned for receiving a liquid processing reagent, and, optionally comprises a cylindrical portion for receiving a swab containing a patient sample.

[0053] The immunoassay test strip comprises a matrix having a sample receiving zone for receiving the processing reagent liquid containing the treated sample suspected of comprising infectious pathogen antigen, a labeling zone having a polyclonal antibody for specifically labeling the antigen as it passes there through and a capture zone having means for specifically binding the labeled antigen thereon, wherein the sample receiving zone, the labeling zone and the capture zone are arranged on the matrix in a liquid flow path. In some embodiments, polyclonal antibodies are coupled with microbeads (europium chelate-impregnated microbeads, or colored or magnetic microbeads) or gold sol, or carbon microparticles. [0054] In some embodiments, the biological sample is collected with a pharyngeal swab. In some embodiments, the biological sample is collected with a swab of the pharynx, tongue, cheek, teeth, gums or nasal passages. In some embodiments, a body fluid is sampled, such as urine, saliva, sputum, mucous, blood, blood components such as plasma or serum, amniotic fluid, semen, wound secretions, vaginal secretions, tears, spinal fluid, washings, and other bodily fluids. Included among the sample are swab specimens from, e.g., the cervix, urethra, nostril, and throat.

Kits

[0055] Kits comprising an assay as described herein are also contemplated. In one embodiment, the kit comprises (i) an immunoassay device or a container with reagents, such as primers and probes, for detection of infectious pathogen antigen (ii) a processing reagent for reducing and/or eliminating infectivity of the biological sample, and (iii) an illumination source or instructions for use of such illumination source. The kits may additionally include any one or more of written instructions for using the device or container and/or collecting a biological sample, an instrument or tool for collecting a biological sample, labels for marking the device or container, and other containers or vials containing a reagent for preparing a treated sample. The kits may additionally include instructions for reading and interpreting the results of an assay. The kits may further comprise reference samples that may be used to compare test results with the specimen samples. In one embodiment the kits include a swab for collecting a biological sample, and instructions for use of the assay and for collecting the sample.

EXAMPLES

[0056] The following examples describe exemplary assays that can be performed using the presently disclosed methods and compositions. However, the present disclosure shall in no way be considered to be limited to the particular embodiments described below.

EXAMPLE 1: DETECTION OF INACTIVATED SARS COV-2

[0057] Infectious biological samples containing SARS CoV-2 virus were inactivated to reduce infectivity by three different inactivation methods. Without being bound by theory, it is believed that all inactivation methods result in production of samples that no longer infective. However, it is also believed that each inactivation method is associated with varying amounts of destruction of viral material and biological damage which may be of consequence to subsequent analyses.

[0058] The first viral inactivation method utilized the photochemical technology (PCT) inactivation as described herein, wherein the SARS CoV-2 sample was incubated with a processing agent comprising amotosalen (an intercalating compound) and then exposed to UVA (320-400 nm) light at 3 J/s ² for 3-5 minutes.

[0059] For comparison, a second SARS CoV-2 sample was inactivated by heat inactivation and a third SARS CoV-2 sample was inactivated with gamma irradiation.

[0060] The three different inactivated SARS CoV-2 samples were then analyzed for SARS CoV- 2 antigen detection by commercially available instruments (e.g., SOFIA®2 and LYRA®, from Quidel Corporation) and by a portable unit with a camera (PUC).

[0061] Inactivated SARS CoV-2 samples were diluted and tested at concentrations of 2.65xl0 ⁴ , 2.65xl0 ³ , 2.65xl0 ² , 2.65x1b ¹ , 2.65 , 0.27, 0 pfu/mL. Virus dilutions were prepared with saline and 300 pL of each dilution was then added to an extraction reagent (11.7 mM Tris, 28 mM EDTA, 0.08% empigen, 0.023 mg/ml mouse IgG, and 1.4 mM TCEP) tube, mixed and incubated for Imin. Two lateral flow immunoassay cassettes for SARS CoV-2 detection were loaded with 120 pL of inactivated, diluted SARS CoV-2 sample for each concentration. The cassettes were then read by a SOFIA®2 instrument and by the PUC after 15 minutes, in accordance with manufacturer’s instructions. Same virus dilutions were tested on LYRA® as well, in according with manufacturer’s instructions. The results from this analysis, summarized in Table 1, demonstrate that SARS CoV-2 samples inactivated with the PCT method in accordance with the present disclosure include more detectable SARS CoV-2 antigen when analyzed by a SOFIA®2 instrument and by a PUC as compared to heat-inactivated and gamma-irradiated samples (See Table 1 at 2.65 x 10 virus pfu/mL dilution for the immunological assays (SOFIA®2 and PUC) and the 0.27 virus pfu/mL dilution for the nucleic acid-based LYRA® assay).

Table 1: Detection of SARS CoV-2 in Inactivated Samples

[0062] The results demonstrate that samples containing infectious pathogens, such as SARS CoV-2 virus, contain more detectable SARS CoV-2 antigen after inactivation of infectivity by PCT than after inactivation by heat or radiation. Accordingly, the present disclosure provides samples that are both safe to handle and amenable to sensitive and specific diagnostic testing subsequent to reduction of infectivity, due to the largely intact diagnostic signal after inactivation with PCT.

EXAMPLE 2: DETECTION OF SARS COV-2 FROM INFECTIOUS SAMPLES AND PCT INACTIVATED

SAMPLES

[0063] Infectious biological samples containing SARS CoV-2 virus were inactivated by PCT inactivation methods described herein. Next, detection of SARS CoV-2 was performed with SOFIA®2 for the PCT inactivated sample compared to a positive control (untreated, infectious) SARS CoV-2 sample.

[0064] The viral inactivation method utilized photochemical technology (PCT) inactivation as described herein, wherein the SARS CoV-2 sample was incubated with a processing agent comprising amotosalen (an intercalating compound) and then exposed to UVA (320-400 nm) light at 3 J/s ² for 3-5 minutes.

[0065] The inactivated SARS CoV-2 sample and an untreated positive control containing infectious SARS CoV-2 virus were then analyzed for the presence of SARS CoV-2 antigen by the commercially available SOFIA®2 instrument (Quidel Corporation), as described above for Example 1.

[0066] The results from this analysis are shown in Figure 2 that plots amount of virus (log pfue) versus signal/cut-off (S/CO) as detected by SOFIA®2. The results demonstrate that SARS CoV- 2 samples inactivated with the PCT method in accordance with the present disclosure include essentially the same amount of detectable SARS CoV-2 antigen as compared to an untreated, infectious positive control sample (superimposable data points).

[0067] Accordingly, the present disclosure provides samples that are both safe to handle and amenable to sensitive and specific diagnostic testing subsequent to reduction of infectivity, due to the presence of intact diagnostic signal that persists after inactivation by the PCT methods provided herein.

[0068] While various specific embodiments have been illustrated and described, skilled artisans will recognize various modifications, permutations, additions and sub-combinations thereof, and will appreciate that these can be made without departing from the spirit and scope of the present disclosure. Therefore, it is to be understood that the disclosure is not to be limited to the specific embodiments disclosed herein, as such are presented by way of example. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0069] All literature and similar materials cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, internet web pages and other publications cited in the present disclosure, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose to the same extent as if each were individually indicated to be incorporated by reference. In the event that one or more of the incorporated literature and similar materials differs from or contradicts the present disclosure, including, but not limited to defined terms, term usage, described techniques, or the like, the present disclosure controls.