PLURALITY OF DNA SAMPLES

FIELD OF THE INVENTION

The present disclosure relates to means and devices for DNA analysis of the samples, in particular PCR (Polymerase Chain Reaction) analysis.

PRIOR ART

PCR (Polymerase Chain Reaction) is a method that enables the amplification of a specific DNA segment, which can then be used for various studies. The method is made possible by the ability of certain polymerases to stay stable and retain their biological function at high temperatures. Isothermal amplification is an alternative to the PCR method and it can be carried out at a constant temperature, unlike the aforementioned method which cycles between certain temperature set points.

The concentration of DNA found in a specimen sample is too low for it to be used for analysis, this concentration can be even lower depending on the conditions that the specimen was exposed to, age of the specimen or from where it was sampled. Low concentration of DNA can’t be visualized using techniques such as electrophoresis and DNA sequencing, making DNA amplification a necessary step before using the previously mentioned methods.

Electrophoresis is a method used for DNA analysis. The method works on the fact that DNA is slightly negatively charged. An anode and cathode are used for generating an electrical field, considering the negative charge that DNA carries it will move towards the cathode. DNA fragments move through a microporous gel, where bigger fragments travel slower than smaller ones. This leads to forming of dark DNA bands that can be seen under UV light.

DNA sequencing is a method used in DNA analysis which shows the nucleotide order of that segment. During PCR amplification, special nucleotides that have specific fluorescent markers (nucleotides A, G, C, T, have different fluorescent markers) are used and when they bind, they stop further amplification of that segment. Considering the amount of DNA copies that are produced during PCR, there is a 100% chance that amplification will be stopped at every nucleotide in the DNA segment that is being amplified. These amplified segments, of various lengths, are then put through a microcapillary tube. The tube is filled with a microporous gel, where shorter segments travel faster than longer ones. A laser beam excites the fluorescent markers, which is picked up by a photosensor. This data is then processed by a specialized software that will show the order of nucleotides in the wanted DNA segment.

In most cases, in a PCR protocol there are five phases: primary denaturation, denaturation, annealing, extension and final extension. These phases differ in their set temperatures. Primary denaturation and denaturation are steps that require high temperatures (in most cases between 92°- 98°C), annealing is done at temperatures that mostly range from 50°-60°C, extension and final extension are almost always performed at a temperature of 72°C. These phases are repeated cyclically, over any given number of cycles.

Prior art devices include tools such as educational tools, which yet do not include integrated functions for changing the protocol, and require the usage of software on a computer by connecting an analysis device to a PC/laptop, and the subsequent uploading of protocol to the device. Such devices known in the art also do not include integrated batteries, do not use resort to controllable heating/cooling means and have small response capacities. Furthermore, such devices use standard PCR tubes, not allowing that reactions are done directly in disposable well plates.

In the article „Portable and Battery-Powered PCR Device for DNA Amplification and Fluorescence Detection", Sensors 2020, 20, 2627, Junyao Jie et al. disclose a battery-powered, portable PCR device for PCR amplification and end-point detection. The device consists of a PCR thermal control system, PCR reaction chip and a detection system. Further the device comprises thin-film heaters and metallic RTDs. Said reaction chip contains four thin-film heaters and four RTDs. The reaction chips are made of a silicon substrate and being disposed to the PCR thermal control chip.

DNA analysis is used in a wide spectrum of professions, some of which are: biology, forensic science, medicine, pathology, veterinary science, pharmaceutics, ecological studies etc. This makes the PCR method essential for these professions.

Biologists and ecologists that are regularly out in the field have to take samples, which can range from hole insects to pond water. These samples have to be packed and adequately preserved, before they are sent to a laboratory for further testing. Otherwise, degradation will occur which will lower the overall concentration of DNA. Depending on the level of DNA degradation, it can lower the quality of the results from further analysis or even make the samples unviable.

With a portable DNA analysis device as that of the present disclosure, there would be no need for sample preservation. The researcher can complete DNA amplification while on terrain, in which case they can use other commercially available devices for further DNA analysis, or on their way back to the laboratory. No preservation means less costs, also it will save them time because there is no need for them to come back to the laboratory to begin with the process of sample testing. But the biggest benefit of being able to amplify DNA on terrain is that they don’t have to worry about quality of the results and they can consistently get results of the same level of quality.

Death of livestock, during its transport between countries, causes major problems. Veterinary forensic investigators have to determine the exact cause of death within a short time frame (in most cases 72h), so that the insurance can be claimed on the dead animal. Also, these limitations are set in place so that if the diseases, from which the livestock died, aren’t native for that region they can react in a timely manner and stop it from spreading to animals that are living in that region. Considering that the place where the animal died and where their laboratory is located can be hours away from each other, speeding up the diagnostic process is of most importance. The solution of the present disclosure can be of crucial help here, it will enable the veterinary forensic investigator to start the diagnostic process while still on the scene. This will speed up the diagnostic process in the conditions where time is a limiting factor.

Countries in less developed, rural areas don’t have a developed, accessible healthcare system. These countries are often ravaged by diseases such as malaria, cholera, hepatitis etc. People infected with such diseases are often neglected and don’t seek needed medical help and diagnosis. Even if they had the means to get the proper diagnosis it is uncertain that the medical system there has the tools needed for it.

The solution of the present disclosure can provide these vulnerable groups much needed medical aid. The solution of the present disclosure can thus be used for diagnosing a variety of diseases without the need of going to a hospital or having a specialized laboratory. It could be used in point-of-care testing in the field, bringing the medical care to the vulnerable groups. This could be vital in early prevention of diseases outbreaks as well as bettering the general health of the country’s population.

SUMMARY OF THE INVENTION

The present disclosure comprises a plate for the analysis of a plurality of DNA samples. The plate may comprise a plurality of wells, each well being suitable for providing an individual DNA sample, wherein the plate comprises a metal which is able to be heated through induction heating, in particular the plate being such that the wells are able to be heated through induction heating provided on a side of the plate opposite to the wells.

The metal may comprise or consist of steel. The metal may comprise or consist of stainless steel.

The wells may have a capacity for receiving a DNA sample which is of 20-70 pl, optionally 30- 60 pl, optionally 45-55 pl.

The plate may comprise 64-625 wells, optionally 100-400 wells, optionally 289-361 wells. The wells may have a form which provides capillary forces in a sample provided in a well. The form of the wells may be substantially cylindrical or substantially conical.

The present disclosure may further comprise an assembly for the analysis of a plurality of DNA samples. The assembly may comprise a plate according to the present disclosure and a plurality of lids, each lid covering a well.

The lids may comprise a metal which is not able to be heated through induction heating. The metal of the lids may comprise or consist of copper.

The present disclosure may further comprise an apparatus for the analysis of a plurality of DNA samples. The apparatus may comprise a plate according to the present disclosure or an assembly according to the present disclosure. The apparatus may further comprise a bed for providing the plate and induction heating means, the induction heating means being arranged adjacently to the bed such that they induction heat a side of the plate opposite to the wells, the bed optionally being configured to removably fix the plate.

The apparatus may comprise at least one controller, the controller being configured to control the heating action of the induction heating means to increase or maintain the temperature of samples provided in the wells.

The apparatus may further comprise cooling means, the cooling means being arranged adjacently to the bed such that that they provide the cooling of the plate and respective wells. The controller may be further configured to control the cooling action of the cooling means to lower or maintain the temperature of samples provided in the wells.

The controller may be further configured to control the heating action of the induction heating means and the cooling action of the cooling means such that, through such control, the temperature of samples provided in the plate is increased, decreased and/or maintained, thereby implementing a DNA analysis protocol.

The said configuration of the controller to increase, decrease and/or maintain the temperature of the samples in the plate may be such that the DNA analysis protocol comprises the phases of: a) primary denaturation, b) denaturation, c) annealing, d) extension, and e) final extension, of the samples provided in the plate.

The said configuration of the controller to increase, decrease and/or maintain the temperature of the samples in the plate may be such that the DNA analysis protocol comprises the phases of: a) primary denaturation, wherein primary denaturation comprises: i) increasing the temperature until a primary denaturation temperature is substantially reached, and ii) maintaining a primary denaturation temperature while a primary denaturation time has not elapsed, b) denaturation, wherein denaturation comprises: i) decreasing or increasing temperature until a denaturation temperature is substantially reached, and ii) maintaining a denaturation temperature while a denaturation time has not elapsed, c) annealing, wherein annealing comprises: i) decreasing or increasing temperature until an annealing temperature is substantially reached, and ii) maintaining an annealing temperature while an annealing time has not elapsed, d) extension, wherein extension comprises: i) decreasing or increasing temperature until an extension temperature is substantially reached, and ii) maintaining an extension temperature while an extension time has not elapsed, and/or e) final extension, wherein final extension comprises i) decreasing or increasing temperature until a final extension temperature is substantially reached, and ii) maintaining a final extension temperature while a final extension time has not elapsed.

Moreover, the apparatus may comprise one or more temperature sensors. The temperature sensors are provided such that the temperature in wells is determined and or deduced. At least one temperature data may be provided to the controller, such that it controls the induction heating means and/or the cooling means to increase, decrease or maintain the temperature.

DESCRIPTION OF FIGURES

The present disclosure is described in more detail hereinafter with reference to the accompanying drawings.

Figure 1 shows a top - Figure 1A - and a side view - Figure IB - of well plate 361 (1.1) according to the one embodiment of the present disclosure.

Figure 2 shows a dimetric view of well plate 361 (1.1) according to further embodiment of the present disclosure.

Figure 3 shows a top view of well plate 100 (1.2) according to another embodiment of the present disclosure.

Figure 4 shows a top view of well plate 36 (1.3).

Figure 5 shows a top view of the well plate holding compartment.

Figure 6 shows Default PCR protocol phases.

Figure 7 shows Internal states for default PCR protocol phases.

Figure 8 represents logic used for alternating between phases of default PCR protocol.

DETAILED DESCRIPTION

As previously described, the present disclosure comprises a plate for the analysis of a plurality of DNA samples, the plate comprising a plurality of wells, each well being suitable for providing an individual DNA sample. The plate comprises a metal which is able to be heated through induction heating, in particular the plate being such that the wells are able to be heated through induction heating provided on a side of the plate opposite to the wells. The said metal allows the usage of induction heating to heat the samples. Such provision has the advantage that induction heating means may be used. Such means are easily controllable, thereby permitting the usage of a controller which implements a DNA analysis protocol. The plate of the present disclosure thereby enables a reduced size device, as well as an application in various non- laboratory conditions, not requiring the use of plastic tubes and thus leading to a reduction in plastic waste.

A particularly suitable metal for such purpose is steel. Stainless steel allows to reuse the plate. As referred, the wells may have a capacity for receiving a DNA sample which is of 20-70 pl, optionally 30-60 pl, optionally 45-55 pl.

The plate may have different sizes, suitable for different applications, in particular 64-625 wells, optionally 100-400 wells, optionally 289-361 wells.

The wells may have a form which provides capillary forces in a sample provided in a well. Capillary forces within the wells are high such that it prevents the liquid from spilling out and cross-contamination from occurring. Steel well plates are intended for single use only, and after use will be recycled into material for new well plates. Wells may be made from stainless steel and not be single use only. With sterilization methods effective in washing off the contaminants from well plates it will allow for multiple use of a single well plate.

The form of the wells may be substantially cylindrical or substantially conical. The diameter, depth and therefore the volume, and the raster of the wells is suitable to change. Shape of the wells also may change from cylindrical to conical.

As referred, the present disclosure may further comprise an assembly for the analysis of a plurality of DNA samples. The assembly may comprise a plate according to the present disclosure and a plurality of lids, each lid covering a well.

The lids may comprise a metal which is not able to be heated through induction heating. The metal of the lids may comprise or consist of copper. Copper is an exemplary material which may be used as it isn’t affected by the induction heating effect. Each lid may be defined as matching in size the respective well.

All wells may have the same size and form.

All wells may be equally apart from each other.

As referred, the present disclosure may further comprise an apparatus for the analysis of a plurality of DNA samples. The apparatus may comprise a plate according to the present disclosure or an assembly according to the present disclosure. The apparatus may further comprise a bed for providing the plate and induction heating means, the induction heating means being arranged adjacently to the bed such that they induction heat a side of the plate opposite to the wells, the bed optionally being configured to removably fix the plate.

As referred, induction heating means provide an advantage, as such means are easily controllable, thereby permitting the usage of a controller which implements a DNA analysis protocol. The apparatus of the present disclosure thereby enables a reduced size device. The low consumption of such heating means also enables the powering of the device through a battery, in which case the apparatus comprises a battery.

As referred, the apparatus may comprise at least one controller, the controller being configured to control the heating action of the induction heating means to increase or maintain the temperature of samples provided in the wells. The controller may comprise one or more microcontrollers, which control the operation of the induction heating means. Such control may comprise turning the induction heating means on or off. It may also comprise adjusting the power of the induction heating means.

Moreover, the apparatus may further comprise cooling means, the cooling means being arranged adjacently to the bed such that that they provide the cooling of the plate and respective wells.

As referred, the controller may be further configured to control the cooling action of the cooling means to lower or maintain the temperature of samples provided in the wells. Such control may comprise turning the cooling means on or off. It may also comprise adjusting the power of the induction heating means.

The controller may be further configured to control the heating action of the induction heating means and the cooling action of the cooling means such that, through such control, the temperature of samples provided in the plate is increased, decreased and/or maintained, thereby implementing a DNA analysis protocol.

The said configuration of the controller to increase, decrease and/or maintain the temperature of the samples in the plate may be such that the DNA analysis protocol comprises the phases of: a) primary denaturation, b) denaturation, c) annealing, d) extension, and e) final extension, of the samples provided in the plate.

As previously referred, such consist of preset stages of a DNA analysis protocol, which are adaptable to each protocol. For instance, to each of said phases may correspond a predefined temperature and time during which the respective predefined temperature is maintained.

The sequence of the phases is as described above. The said configuration of the controller to increase, decrease and/or maintain the temperature of the samples in the plate may be such that the DNA analysis protocol comprises the phases of: a) primary denaturation, wherein primary denaturation comprises: i) increasing the temperature until a primary denaturation temperature is substantially reached, and ii) maintaining a primary denaturation temperature while a primary denaturation time has not elapsed, b) denaturation, wherein denaturation comprises: i) decreasing or increasing temperature until a denaturation temperature is substantially reached, and ii) maintaining a denaturation temperature while a denaturation time has not elapsed, c) annealing, wherein annealing comprises: i) decreasing or increasing temperature until an annealing temperature is substantially reached, and ii) maintaining an annealing temperature while an annealing time has not elapsed, d) extension, wherein extension comprises: i) decreasing or increasing temperature until an extension temperature is substantially reached, and ii) maintaining an extension temperature while an extension time has not elapsed, and e) final extension, wherein final extension comprises i) decreasing or increasing temperature until a final extension temperature is substantially reached, and ii) maintaining a final extension temperature while a final extension time has not elapsed.

The induction heating means and/or the cooling means are controllable by means of a controller. The controller may comprise an adaptable PID loop. PID (Proportional-Integral-Derivative) controller is a control-loop mechanism employing feedback mechanism to control process variables. The PID controller continuously calculates an error e(t) as the difference between a desired setpoint (SP) and a measured process variable (PV) and applies a correction based on proportional, integral, and derivative terms (denoted P, I, and D respectively).

Namely, This PID loop enables the controller to regulate and maintain the set temperatures for each phase via digital signal.

The induction heating means further allow to, when controlled with a PID controller, maintain a certain temperature extremely precisely, showing even 0.00°C deviation from the setpoint. The PID loop is adaptable, meaning it doesn’t need constant conditions for it to stably maintain the set temperature. This enables the apparatus to always maintain the same setpoint, regardless of external factors such as air temperature or humidity. PID also regulates the function of fans that are used to cool down the electrical components.

The plate, the assembly and, in particular, the apparatus of the present disclosure, provide a portable, stand-alone means for DNA amplification.

The apparatus may be configured, for instance through a particular configuration of its controller, perform both the PCR method as well as the Isothermal amplification method.

Several additional and combinable details of the plate, assembly and apparatus of the present disclosure are subsequently described. Subsequently, the terms apparatus and device are interchangeably used to refer to the apparatus of the present disclosure.

The apparatus of the present disclosure is stand-alone in the manner that it may its own power supply and microcontroller(s), meaning it doesn’t depend on external battery packs or software run on a computer or a laptop.

Portability is presented in its compact size and lightweight characteristic, enabled by using Li- ion battery. The apparatus may be able perform up to four hundred (400) reactions on its unique well-plate that doesn’t require use of plastic tubes. Namely, and as previously described the apparatus according to present disclosure has disposable, metal well plates in which reactions are performed directly without the use of tubes.

These three key functions of the apparatus make it usable even in non- laboratory conditions, or putting it in other words, it makes the apparatus usable anywhere from a forest to a motel-room. This means that the apparatus can make DNA amplification available anywhere which will greatly improve workflow of various professions that use DNA amplification methods on a daily basis.

As discussed above, there is a strong need for apparatuses usable even in various, nonlaboratory conditions, not requiring the use of plastic tubes and thus leads to a reduction in plastic waste. The present apparatus enables shortening of the PCR sample preparation process and faster obtaining of the analysis results.

The apparatus of the present disclosure may be a compact, portable device for DNA amplification whose dimensions are 300x300x180mm.

The operating current of the device is 24V, supplied by 24 V, 24.5 Ah Li-ion battery. 24V, 600W medical power supply is used for charging up the battery and as a power source for when the device is plugged in. 32bit microcontroller is used for processing inputs and outputs and for the control of the device. A touch screen display may show all the needed parameters, such as: current temperature, set point, current phase, cycle number and an estimated time in which the protocol will be finished. Touch screen also enables direct data entry for the changing variables in the protocol, such as: temperature of specific phase, duration of each phase, number of cycles. The plate of the present disclosure has a unique design, as previously referred. It allows to reduce plastic waste and shorten the procedure of PCR sample preparation. They are made of 6mm steel plates, steel must be used for the induction heater to work on them. Each plate on its top side has cylindrical holes, or wells, that are 4mm in diameter and 4mm deep which gives them a total volume of 50.27pL (volume of cylinder is calculated by formula: V=7rr ² h). This is sufficient for most reactions, considering that a typical reaction has a 30pL volume.

Three types of wells may be used, differing in their reaction capacity. The biggest well plate (1.1) provides 361 reactions (Figure 1), with dimensions of 100x100x6mm (WxLxH) and a 19x19 hole raster (Figure 2). This number of reactions isn’t used daily, and for that reason there is also 100 reaction capacity well plate (1.2) represented in Figure 3 and 36 reaction capacity well plate (1.3) represented in Figure 4. Capillary forces within the wells are so high that it prevents the liquid from spilling out and cross-contamination from occurring. Steel well plates are intended for single use only, and after use will be recycled into material for new well plates. Each well plate type has its own copper lid, matching in size. Copper is used as it isn’t affected by the induction heating effect.

The diameter, depth and therefore the volume, and the raster of the wells is suitable to change. Shape of the wells also may change from cylindrical to conical. Also, well plates might be made from stainless steel and not be single use only. With sterilization methods which are effective in washing off the contaminants from well plates it will allow for multiple use of a single well plate.

Single use well plates are placed in a special compartment and locked into place. The bed of this compartment, on which the well plate is placed, is 120x120mm in size and it has a bracket (distance between bracket pins is 100mm) that accepts well plate 361 (Figure 5). Other well plates have a special plastic frame in which they are put so that they can be locked into place by the bracket. On the bottom of the bed thermal sensors may be placed, for measuring the temperature of the well plate.

Underneath this compartment the induction heating means and the cooling means may be provided. Such combination of means may be called a heating/cooling unit is located. The cooling part of the unit may consist of a special canal, which connects to the other side of the bed that also has special air slits that allow the air to pass and cool down the well plate. On the other end of the canal may be provided a high airflow fan, with dimension of 120x120x25mm, pumping air into the canal, which channels it towards the plate. The heating component of the unit may consist of a planar, PCB induction heater, with dimensions of 100x100mm and a maximum power of 150W at 24V.

Additional parts may be added to the cooling unit, in the form of a TEC element and copper heatsink (or some other unit that will aid in better cold temperature transfer). Addition of these parts allows the apparatus of the present disclosure to reach temperatures lower than ambient temperature and speed up the cooling process.

Both the cooling and the heating element may be controlled with an adaptable PID loop. PID (Proportional-Integral-Derivative) controller is a control-loop mechanism employing feedback mechanism to control process variables. PID controller continuously calculates an error e(t) as the difference between a desired setpoint (SP) and a measured process variable (PV) and applies a correction based on proportional, integral, and derivative terms (denoted P, I, and D respectively). Namely, This PID loop enables the uC to regulate and maintain the set temperatures for each phase via digital signal. Induction heater is chosen because when controlled with a PID controller it will maintain a certain temperature extremely precisely, showing even 0.00°C deviation from the setpoint. PID loop is adaptable, meaning it doesn’t need constant conditions for it to stably maintain the set temperature. This enables the device to always maintain the same setpoint, regardless of external factors such as air temperature or humidity. PID also regulates the function of fans that are used to cool down the electrical components.

A top heater may be used to prevent condensation, from heating of the well plate, to occur inside the wells. Planar resistance heater, with a maximum power of 100W, is used for this purpose. It is enveloped by a thin piece of copper sheet, and lays on top of the well plate cover. Top heater should maintain a temperature above 100°C, most often 105°C is used. Temperature regulation is done with PID controller. Electronics of the device according to the present disclosure may have a modular design. It is powered by a 24V, 600W medical grade power supply and a 24V, 24.5 Ah Li-ion battery. The battery allows the device to work on the field independent from a power grid. On the other hand, the power supply is used for charging the battery and for powering the device when it is connected to utility power.

The electronics enable fast and very precise PID temperature control. Thanks to a fast processor and ICs, the closed circuit provides a resolution that gives almost linear PID control.

Adaptable PID routine is used which enables constant and reproducible outcomes in maintaining the temperature setpoint, and doesn’t require constant environmental conditions.

Temperature sensors may consist of PtlOO probes. Such probes are used for measuring the temperature of the well plate, because they have a fast response time and aren’t affected by the magnetic field generated by the induction heater. The slower response NTC probes are used for monitoring the ambient temperature. These two elements provide the key inputs for PID controller: Ptl 00 probes for adjusting the output so that the setpoint can be reached quickly and maintained with a high degree of precision, and NTC probes for adjusting the PID outputs in accordance to outside temperature conditions. PID routine is called out for controlling the top heater, induction heater as well as the fans.

The apparatus may further comprise communication means. The communication means may be cabled or wireless. The communication means may be CANbus or RS232 compatible.

The controller may be configured with a logic of alternating temperatures according to phases in a PCR protocol. A default PCR protocol may comprise the following phases:

1) Primary Denaturation,

2) Denaturation,

3) Annealing,

4) Extension,

5) Final Extension.

The concrete temperature for each phase may be defined according to a particular protocol. For each protocol, the temperature alternating logic will alternate through phases as represented in the Figure 6.

Protocol phases depicted in the Figure 6 were mapped to internal states which are represented in the Figure 7.

Based on the current internal state of the protocol, temperature is either rising, falling or being maintained in order to reach the next internal state. When all states are finished, the protocol itself is finished. The steps performed based on the current internal state are represented in the Figure 8.

The configuration of the controller allows modification of protocol phases in a way that it supports creating custom protocol with custom phases with custom lengths.

The apparatus, the system, the method and/or their elements include components to perform at least some of the example features and features of the methods described, whether through hardware components (such as memory and / or processor), software or any combination thereof.

In particular, the apparatus and/or the controller may comprise one or more microcontrollers, such as the segment and master microcontrollers, with the capability of receiving external data through inputs and providing actuation of outputs, based on predefined configured rules and/or programming.

An article for use with the apparatus, the controller, respective methods and/or their elements, such as a pre-recorded storage device or other similar computer-readable medium, including program instructions recorded on it, or a computer data signal carrying readable program instructions computer can direct a device to facilitate the implementation of the methods described herein. It is understood that such apparatus, articles of manufacture and computer data signals are also within the scope of the present disclosure.

A "computer-readable medium" means any medium that can store instructions for use or execution by a computer or other computing device, including read-only memory (ROM), erasable programmable read-only memory (EPROM) or flash memory, random access memory (RAM), a portable floppy disk, a drive hard drive (HDD), a solid state storage device (for example, NAND flash or synchronous dynamic RAM (SDRAM)), and/or an optical disc such as a Compact Disc (CD), Digital Versatile Disc (DVD) or Blu-Ray ™ Disc.

As will be clear to one skilled in the art, the present disclosure should not be limited to the specific details described herein, and a number of changes are possible which remain within the scope of the present disclosure.

Of course, details of the plate, the assembly and/or the apparatus shown above are combinable, in the different possible forms, being herein avoided the repetition all such combinations.