System and method of nucleic acid amplification for point of collection

Abstract

A system for nucleic acid amplification is to synthesize amplified target nucleic acids or determine the presence of target nucleic acid. The mobile device of the system may be implemented with software for analyzing the reaction or optionally delivering the information of a sample to a cloud. Therefore, the system can provide corresponding genetic information of organism, cancer cells or viruses of interest. The information may include gene expression levels of interest, DNA identity of samples as well as treatment suggestion and professional lists for consulting. The system could also optionally be used with a mobile device to amplify the target nucleic acid for the downstream sequencing or measurement.

Claims

1. A system for processing a sample, the system comprising: at least one heat source; at least one reaction chamber on a test platform; nucleic acid amplification reaction reagents reacting with said sample through nucleic acid amplification reactions to produce amplified nucleic acid; a mobile device having a detection module and installed software; and means to shuttle said reaction chamber to a position; wherein said position corresponds to either the proximity of said at least one heat source or a measurement position for said nucleic acid amplification reactions; wherein said measurement position is suitable for said detection module to perform measurement or collect amplified nucleic acid for measurement; wherein said measurement is either to take an image of said nucleic acid amplification reactions or to sequence said amplified nucleic acid; wherein said mobile device is configured in a manner to quantify and/or sequence said amplified nucleic acid; wherein said at least one heat source has thermal communication with said at least one reaction chamber; wherein said means shuttles said at least one reaction chamber to control the temperature and duration of said nucleic acid amplification reaction, or to a suitable position for quantifying and/or sequencing said amplified nucleic acid via said mobile device; wherein said installed software processes the image taken by said detection module or sequencing data generated by said detection module.

2. The system of claim 1, wherein said at least one heat source comprises either a chemically activated heating material or a container with an electric thermal stat.

3. The system of claim 1, wherein said means may be a combination of one or more of arms, linkages, belts or similar facilities that cause said at least one reaction chamber to have thermal communication with at least one heat source.

4. The system of claim 1, wherein said detection module is a nanopore sequencer.

5. The system of claim 1, wherein said installed software implemented a t-test method or analysis of variance method to determine whether samples in a control group are different from samples in a treatment group within a confidential level.

6. The system of claim 1, wherein said detection module is a camera of said mobile device.

7. The system of claim 1, wherein said at least one reaction chamber is a capillary with at least one closed end.

8. The system of claim 1, wherein said detection module comprises an LED light source.

9. The system of claim 1, wherein said test platform comprises a color calibration or a temperature label.

10. The system of claim 1, wherein said nucleic acid amplification reaction reagents are either lyophilized or stored in a wax.

11. A method for processing a sample, the method comprising the steps of: providing (i) a test platform that includes at least one reaction chamber to receive said sample and nucleic acid amplification reaction reagents, wherein said sample and said nucleic acid amplification reaction agents cause nucleic acid amplification reaction to produce amplified nucleic acid; (ii) a plurality of heat sources; (iii) at least one mobile device with a detection module for said amplified nucleic acid; (iv) means to shuttle said at least one reaction chamber to different positions; wherein said positions are adjacent to either said plurality of heat sources or other positions suitable for taking an image of said nucleic acid amplification reaction or collection of said amplified nucleic acid; introducing said sample into said at least one reaction chamber; sealing said at least one reaction chamber; controlling the temperature of said reaction chamber for said nucleic acid amplification reaction via shuttling said reaction chamber to the proximity of said plurality of heat sources or between proximity of said plurality of heat sources and said detection module, wherein said plurality of heat sources each has a particular temperature, wherein said plurality of heat sources has thermal communication with said at least one reaction chamber when said at least one reaction chamber is adjacent to said plurality of heat sources, thereby the temperature of said at least one reaction chamber is controlled by moving said at least one reaction chamber adjacent to one of said plurality of heat sources having a particular temperature, wherein a duration of said temperature of said at least one reaction chamber is controlled by holding said at least one reaction chamber adjacent to said one of the plurality of heat sources for a specific period of time; and detecting said amplified nucleic acid via shuttling said reaction chamber to a suitable position for said mobile device to take at least one image of said nucleic acid amplification reaction or sequencing said amplified nucleic acid via shuttling said reaction chamber to a suitable position for collection of said amplified nucleic acid for sequencing by said detection module of said mobile device.

12. The method of claim 11, wherein said image is used to quantify said nucleic acid amplification reaction with colorimetric method.

13. The method of claim 11, wherein said sample is prepared with one of said plurality of heat sources.

14. The method of claim 11, wherein said mobile device may transmit said image or said sequencing result to a cloud device.

15. The method of claim 11, wherein the step of sealing said at least one reaction chamber is performed with a grease, wax, or sealant.

16. The method of claim 11, wherein the step of introducing said sample into said at least one reaction chamber is performed through at least one capillary via either capillary action or suction of a suction device.

17. The method of claim 11, wherein said nucleic acid amplification reaction is a polymerase chain reaction or nucleic isothermal amplification reaction.

18. A method for processing a plurality of nucleic acid amplification reactions in a point-of-collection manner, the method comprising the steps of: providing (i) at least one heat source; (ii) at least one receptacle to accommodate the plurality of nucleic acid amplification reactions; (iii) a mobile device having a detection module; (vi) one or more test subjects; and (v) means to shuttle said at least one receptacle; introducing nucleic acid from said one or more test subjects; shuttling said at least one receptacle to the proximity of said at least one heat source to cause said plurality of nucleic acid amplification reactions to complete; shuttling said at least one receptacle to the proximity of said detection module for a suitable position for quantifying amplified nucleic acids produced from said plurality of nucleic acid amplification reactions; quantifying said amplified nucleic acids to obtain a plurality of nucleic acid amplification reaction results; performing analysis of said plurality of nucleic acid amplification reaction results; and reporting said results on said mobile device or transmit said results to a cloud device.

19. The method of claim 18, wherein said plurality of nucleic acid amplification reactions use different primer sets for different genome locations of the same test subject.

20. The method of claim 18, wherein said plurality of nucleic acid amplification reactions use one identical primer set for identical genome locations of a test subject.

Description

BRIEF DESCRIPTION OF DRAWING

[0125] FIG. 1 illustrates a portable nucleic acid amplification system;

[0126] FIG. 2 illustrates a carrier which has four wheels and powered by a battery as a device for nucleic acid amplification;

[0127] FIG. 3 illustrates a receptacle. The receptacle can accommodate plurality of nucleic acid reactions, and has a thin and flat bottom surface as well;

[0128] FIG. 4 illustrate a chemically powered heat source comprises of a thermo, a chemically activated heating material, a aluminum foil cup, oil with low evaporation rate, water, and lid for the thermo;

[0129] FIG. 5 illustrates a receptacle accommodates plurality of capillaries, which can be a mean for transfer reagents or samples. Or the capillaries can serve as reaction chambers;

[0130] FIG. 6 illustrates the steps of a method for obtaining a point-of-collection, selected quantitative indicia of a sample on a test platform, according to an embodiment of the invention;

[0131] FIG. 7 is a high-level flow chart expressing the steps of a method for measuring a target of a nucleic acid amplification reaction in a biological sample using a mobile device according to an embodiment of the invention;

[0132] FIG. 8 is a flow chart of an embodiment method in terms of operational steps, procedures, reaction stages;

[0133] FIG. 9 is a mobile device adapted with a nanopore sequencer for nucleic acid sequencing;

[0134] FIG. 10 is a histogram of Hue values obtained from control and treatment samples;

[0135] FIG. 11 illustrates a portable nucleic acid amplification system with one heat source; and

[0136] FIG. 12 illustrates: a system of nucleic acid amplification with a mobile device using a tourbillion as means for driving the reaction chamber and controlling temperature of reaction.

[0137] While the present invention has been described above in terms of specific embodiments, it is to be understood that the invention is not limited to these disclosed embodiments. Many modifications and other embodiments of the invention will come to mind of those skilled in the art to which this invention pertains, and which are intended to be and are covered by both this disclosure and the appended claims.

[0138] It is indeed intended that the scope of the invention should be determined by proper interpretation and construction of the appended claims and their legal equivalents, as understood by those of skill in the art relying upon the disclosure in this specification and the attached drawings.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0139] As used herein, the term “move relatively” means the translocation between two positions is a motion between two positions in a system.

[0140] As used herein, the term “mobile device” means a mobile apparatus (or handheld computer) that is capable of running a programmed application suitable for executing the embodied functionality. It is a computer small enough to hold and operate in the hand. While suitable traditional smart phones may include products such as, e.g., the iPhone, iPad (Apple, Inc.), Android-based devices, Windows, HarmonyOS-based device and other well known devices and associated operating systems, the term mobile device as discussed and embodied herein is intended to include any digital mobile device such as smartphones, tablets, phablets, smart watches, mobile computer, digital camera, smart glass and other current or future smartphone platforms having similar minimal functionality.

[0141] In this regard and for the sake of clarity, a laptop computer might be covered under the definitional use of the term mobile device; but not a computing device that could be made portable or mobile by an accompanying apparatus that might give it portability mobility. Thus, the term “mobile device will be used herein (including the claims) to mean devices as discussed within the paragraph above.

[0142] It should be understood that the term “adjacent” (and in the claims) does not require that the reaction chamber be in directly contact with the heat source.

[0143] As used here “driven” shall include any form of drive mechanism or facilities for inducing motion in embodiments. It includes a combination of motor or gears, and the source of driving energy can be one or a combination of electric or mechanic or chemical energy.

[0144] As used here, “arm” shall include a linkage that may include one or more arms or leg members, bearings, and one or more receptacles for holding or gripping reaction chambers.

[0145] The term “colorimetric test platform”, “colorimetric measurement”, or “colorimetric reactive” as may be used herein means at least a measurable color change from one color to a different color or a measurable change in intensity of a particular color, in the presence of nucleic amplification reaction or due to temperature change of labels or reactions.

[0146] The term “suitable” as may be used herein (and in the claims) means having the qualities that are correct, needed, or appropriate for something, especially as a person skilled in the art would understand.

[0147] The term “about” as may be used herein means the amount of the specified quantity plus/minus a fractional amount thereof that a person skilled in the art would recognize as typical and reasonable for that particular quantity or measurement.

[0148] The term “test kits” or “kits” or “test platform” refers to a test platform; or a combination of the reagents required for nucleic acid amplification, a cartridge, a receptacle or reaction chambers for holding or storing said reagents or reaction.

[0149] Practical examples of embodied test platforms or test kit include, but are not limited to, various custom or commercially available test kits for nucleic acid amplification.

[0150] The term “light source” refers to ambient light source or light emitted by LED or light bulb or laser with a range of spectrum from 180 nm to 1064 nm.

[0151] The term “accessory” or “mobile device accessory” refers to a component of the system and the component is releasably coupled to the mobile device.

[0152] The term “indicia” refers to any physical quantity associated with the color coordinate. The physical quantities include but not limits to pyrophosphate concentration, proton concentration of the reaction, free magnesium ion concentration and amplified nucleic acid concentration, dye concentration or any reactants associate with amplified nucleic acid concentration.

[0153] The term “sample” refers to anything containing amplified nucleic acid and/or nucleic acids obtained from a sample of test.

[0154] The term “temperature label” is a material that change its color when the temperature of its contact changes.

[0155] The term “reactive test region” refers to a region of reaction chamber or a test platform, wherein nucleic acid amplification reaction is hold.

[0156] The term “temperature label” is a material that change its color when the temperature of its contact changes.

[0157] The term “reactive test region” refers to one or more of areas: a region of a kit, a cartridge, a reaction chamber, a receptacle, a test platform, wherein nucleic acid amplification reaction is hold.

[0158] The term “suction device” refers to a bulb or pump that can suck air or liquid from a capillary or a reaction chamber.

[0159] The DNA/RNA is extracted by the other component of the system from any fluid of a sample.

[0160] The other component is a nucleic acid extraction kit/module and/or an external device.

[0161] In one embodiment, a sample is collected and nucleic acid of samples is further processed in a reaction vessel.

[0162] In one embodiment, the reaction vessel is a reaction chamber.

[0163] In one of embodiments, the DNA/RNA for nucleic amplification reaction is introduced to a reaction chamber by a sample/reagent dispensing accessory.

[0164] In one embodiment, the sample/reagent dispensing accessory is one or more capillaries.

[0165] In one of embodiments, the test platform comprises a reactive test region or a reaction chamber, wherein the nucleic acid amplification occurs, and the adjacent heat source has heat communication with the reactive test region.

[0166] In one of embodiment, the reactive test region is a receptacle that holds at least one sample and all reagents required for nucleic acid amplification reaction, wherein the reactive test region is of interest area of colorimetric detection.

[0167] In one of embodiments, at least one accessory dispenses required reagents, enzymes, nucleotides, primers and samples into the reactive test region.

[0168] In one embodiment, a kit comprises nucleic acid amplification reagents for PCR or isothermal amplification reaction.

[0169] In one embodiment, the PCR is a convective polymerase chain reaction.

[0170] In one embodiment, nucleic acid amplification reagents includes but not limited to a combination of DNA polymerase and/or reverse transcriptase, nucleotide, reaction buffers, and/or nucleic acid primers for target nucleic acid fragments, and/or control nucleic acid; and sample preparation reagent may include a combination of cell lysis reagents and/or nucleic acid purification reagents

[0171] In one embodiment, the photo images of nucleic acid amplification reaction of a sample could be processed by the software installed on a mobile device. Therefore, the software identifies if a sample contains target nucleic acid sequences by analyzing the images of reaction through its color coordinate.

[0172] In one embodiment, the color coordinates from an image of test region is corrected against the color coordinate from the calibration region on the same image. Thereby the color difference from images taken by different mobile devices for a particular sample is corrected to a suitable range for colorimetric measurements.

[0173] In one embodiment, a lateral flow assay is performed with the amplified nucleic acid as taught in (Rapid One-Pot Detection of SARS-CoV-2 Based on a Lateral Flow Assay in Clinical Samples, Anal Chem. 93(7)3325 (2021)). The change of color lines on the lateral flow device is further determined by a colorimetric method via using a mobile device for the presence of an interested target.

[0174] A temperature label is a material changing its color with temperature. The change in colors is determined by the mobile device via the image of a temperature label. Therefore, the color change of the temperature label is used for monitoring the temperature of a system.

[0175] In one embodiment, the colorimetric-based method mentioned above is used with temperature label to determine the temperature of a system.

[0176] In one embodiment, the system comprises reagents for nucleic acid sequence amplification, a heat source, a PCM, a temperature label.

[0177] In one embodiment, the system comprises a kit for target nucleic acid sequence amplification, a mobile device, a heat source, a PCM, a temperature label and a tag.

[0178] In one embodiment, a tag may be taken into an image for analysis and/or registration of a test; wherein the image of tag is a QR code or 2D barcode.

[0179] In one embodiment, a tag contains information about the kit or samples or/and users including but not limited to the primers, reactants, enzymes, nucleotides, dye molecules, samples, user information, reagent or/and software version, geographic information, credential information.

[0180] In one embodiment, a tag can associate the mobile device with a cloud service.

[0181] In one embodiment, a tag can associate the nucleic acid amplification results and a cloud service.

[0182] In one embodiment, a tag can associate the geographic location where nucleic acid amplification performed or the location of said mobile device.

[0183] In one embodiment, a test platform comprises at least two reaction chambers. Each reaction on the test reaction chamber is associated with a unique tag. The tag is used to associate a reaction with a sample identity and/or amplification primer sets and/or geometry location and/or a time stamp.

[0184] In one embodiment, the test platform may be contained in a container, which has at least one side as being transparent to allow the detection of color change for image acquisition.

[0185] Furthermore, the software of system associates an information platform which not only identifies the samples or gene expression levels of samples but also provides further information for downstream treatment or management.

[0186] In one embodiment, the results of nucleic acid amplification and geographic location information are sent to cloud and the cloud provides recommendation for a user to take action based on the result or analysis.

[0187] In one embodiment, each reaction is collected in a different reactive test region of a container.

[0188] The container or each reaction region associates with a tag. A tag is used to further associate a reaction with a sample or amplification primer sets by the software, which provides convenience for user to operate sample preparation and record registration.

[0189] In one embodiment, the software is used to monitor the reaction conditions of nucleic acid. The conditions include temperature, amount of synthesized DNA, signal intensities with various temperatures or stages.

[0190] In one embodiment, the software can communicate with a heat source for temperature setting with a wire or wirelessly.

[0191] In one embodiment, the heating source can be an electric thermostat container.

[0192] A statistics method is performed to determine the likelihood of true positive result or true negative result.

[0193] In one embodiment, there are three or more than three samples as control samples while there are three or more than three samples as treatment samples.

[0194] In one embodiment, a t-test or ANOVA is performed to determine the confidence level of true positive or true negative result for samples.

[0195] In one embodiment, a p value of is provided to determine the significance level.

[0196] In one embodiment, at least one statistic methods is implemented in the mobile device of the system or on a cloud service which mobile device links to.

[0197] In one embodiment, the camera of a mobile device is used to directly collect images of reactive test region for determining nuclei acid amplification results. In the embodiment, the mobile device serves as a colorimeter by itself.

[0198] In one embodiment, a temperature label can associate with a heat source or a reactive test region, and the temperature label changes color when the temperature of heat source or of reactive test region changes. Thereby, the temperature of a heat source or reactive test region is monitored via images taken by a mobile device. The mobile device may have software installed, and the software can process the images for the color coordinates and determine the temperature of the heat source or reactive test region.

[0199] In one embodiment, quantification of amplification is by counting the sequence reads generated from a nanopore sequencer.

[0200] In one embodiment, nucleic acid amplification reaction agents include but not limited to a primer set for nucleic acid amplification reaction, DNA polymerase, nucleotide, reaction buffer.

[0201] In term of structure, one of the differences of invention from others is an enclosed house for current invention is optional.

[0202] In term of structure, one of the differences of invention from others is the system comprises a heat conductive reaction chamber which allow measuring indica of samples with a colorimetric method with a mobile device, and easy to scale up the number of reactions in a manner of point-of-collection.

[0203] In term of structure, one of the differences of invention from others is using electricity to power a heat source for nucleic acid amplification reaction or drive a reaction chamber is optional. Thereby the invention may be used in a resource limited area.

[0204] In term of structure, one of the differences of invention from others is said system comprises a mean for translocating a test platform or reaction chamber relatively to various position of a system for thermal communication with heat source or taking image with detection module of a mobile device or collection of nucleic acid amplification product.

[0205] In term of structure, one of the differences of invention from others is the detection module of a mobile device may be a nanopore DNA sequencer and/or an image sensor for sequencing or detection of amplified nucleic acid.

[0206] The present invention is directed to provide system and method of nucleic acid amplification in point-of-collection. The system comprises a heat source for facilitating the nucleic acid amplification reaction and a mobile device for measurement of nucleic acid amplification reaction. The measurement may include use of an image sensor for image acquisition and analysis of images or processing the sequencing data from the amplified nucleic acid produced by the system. The software is a method, and used to quantify amplified nucleic acid according to color change on an image taken or process the sequence data.

[0207] Because a thermal cycler requires a bulky system to conduct heat exchange when a large number of samples are required to process at the same time, it usually is difficult to handle more than 400 samples in a point-of-collection manner. In addition, it usually requires different temperatures for nucleic acid amplification and sample preparation or other biochemical reactions. Furthermore, detection of the result of target nucleic acid amplification during reaction or right after complete of reaction is favorable with a simple method such as colorimetric method or nucleic acid sequencing.

[0208] In FIG. 1, the exemplary embodiment shows three electric thermos10 with three different temperatures as three heat sources. Each has different temperature when filled with water. A test platform 20 is supported by the three thermos. On the platform, there is a step motor 30 connected to an arm 40. The arm hangs up a receptacle 50. And the receptacle accommodates a tube 60. The motor is powered by a battery 70. The position and duration of the arm is controlled by a programmed Arduino UNO R3 and UNL2003 board 80. By rotating the arm, the tube may immerse into each different thermos for each particular period of time. Since each thermo may have different temperatures corresponding to different stage of PCR reaction, immersing the tubes into different thermos may change the temperature of nucleic acid amplification, and cause the reaction to enter into different stages: denaturation of DNA, annealing DNA and DNA synthesis. Thereby, a PCR cycle can be complete by shuttling the tubes between the thermos.

[0209] Once the amplification reaction reaches the predesigned cycle number, the arm can move to the position just right above a cell phone 90. The software can take an image by a user or automatically take the pictures. A LED light source 100 may be used. It depends on which dye molecule is used to detect DNA. In one embodiment, the cell phone takes an image when each time the arm moves over the cell phone. Thereby, one may be able to monitor the amount of nucleic acid amplified over time.

[0210] In FIG. 2, the embodiment shows a carrier has a receptacle 140 and wheels 120 driven by step motors 130, the receptacle has a flat bottom surface. The reaction chambers on the receptacle may contact with a hot plate with a preset temperature. The reaction chambers have openings on top and allow dispensing of reagents and samples. The reagent may contain liquid wax to seal the reaction chamber or prevent the evaporation of buffer from the reaction chambers. The carrier can move forward and backward over the hot plates with different temperatures such as 95 deg. C., 68 deg. C. The carrier can move over a hot plate with 95 deg. C. 141 for cell lysis with a specific time. And then, the carrier can further move to the hot plate with 68 deg. C. 142 for LAMP with other specific period. Finally, the carrier moves forward to facilitate imaging taken by a mobile device 151 on a holder 152. The carrier is controlled by an Arduino Uno R3 board 135.

[0211] The carrier can be used for PCR as well as isothermal nucleic acid amplification reaction. Samples and reagents may be dispensed to reaction chambers on the receptacle. The sample preparation may be performed at 95 deg. C. for DNA denaturation. The carrier may further move forward to a hot plate with 55 deg. C. for primer annealing and then move forward to a template with 72 deg. C. for DNA synthesis. Thereby, a PCR cycle can be complete via movement along three hot plates. Finally, the carrier moves to the position, which allows taking an image by a mobile device. The software installed on the mobile device can extract the RGB values from an image of reaction chambers and color calibration regions, mapping the RGB values to an associated hue value from HSI space. The hue vale may associate with a DNA test result or concentration.

[0212] In FIG. 3, the embodiment schematically illustrates an exemplary embodiment of receptacle for a large number of nucleic acid amplification reactions. The receptacle 160 has plurality of reaction chambers 170 with opening on the top. The dimension of each reaction chamber is around 7 mm×7 mm×5 mm with a 5 mm thick of wall. For a silicon rubber heater with 100 cm×60 cm, it can easily accommodate several thousand of reaction chambers or reactions. The lyophilized plurality of primers sets is allocated into each reaction chamber respectively. Each of primer set may target a genome location of one or more organisms or viruses. The genome locations may be conservative or specific regions of genomes for interested organisms or viruses. An example of organisms or viruses for the primer sets is in the table 1.

[0213] In FIG. 4, the embodiment shows an exemplary embodiment of chemically activated heat source. The heater 180 is inside the thermo 190. Above the heater is a foil cup 200 filled with water, and has oil or was on its top 210 to prevent quickly temperature drop. Oil with low evaporation rate at 95 deg. C., chemically inert with water and has lower density than water is suitable. Once activating the heater with adding water, the lid 220 of thermo is close till the predesigned temperature is reached, which can be observed by the temperature labels. One of examples of heaters is assortment of magnesium, iron and salt. Each heat source can have different temperature by the amount of heater and water added.

[0214] Thereby, the chemically activated heat source for a nucleic acid amplification reaction doesn't require electricity. The water in the foil cup may have thermal communication with a reaction chamber on a test platform.

[0215] In FIG. 5, the embodiment schematically illustrates a capillary can be used as reaction chambers or a mean to transfer samples and reagents. One or more capillaries are immobilized on a receptacle, which is connected to a rubber bulb. The capillaries drawn a sample from a reservoir with a sample by capillary action and dispense the samples to receptacles which may contain reagents and buffer for nucleic acid amplification. After proper mixing of reagents and samples, the capillary can further draw the samples and reagents. The bottom end of a capillary may be fixed with fast-acting adhesive such as cyanoacrylate. The top of capillary may be sealed with wax. Thereby, the capillary may serve as a reaction chamber. In one application, saliva may be collected from an animal or person, the saliva may mix with cell lysis buffer in a reservoir and the reservoir is contacted with a heat source to maintain its temperature at 55 deg. C. to allow the cell lysis reaction to proceed. Once the reaction is complete, the capillaries may draw the nucleic acid from the reservoir and dispense into reaction chambers for nucleic acid amplification reactions and colorimetric measurement. Thereby, each capillary may correspond to a particular reaction chamber in the receptacle. The particular reaction chamber may contain a particular primer set which target a particular genome location of an animal or human.

[0216] In FIG. 6, the embodiment schematically illustrates the steps for extract and analysis of an image from a nucleic acid reaction. It starts with collecting image of a nucleic acid amplification reaction 250. RGBA/YUV values are extracted from image 260, and then the values of image are split into test region and calibration region 300. Extract 100×100 pixels from the calibration region 280. From the median/average values obtained from calibration region 290, a mapping function which can convert measured value to a predesigned value 310. Extract 100×100 pixels from test region 270. The mapping function is then used to convert the median/average of measure values from test region 325 to a value against a designated threshold 330. If the value is over a threshold, the reaction successes 350 otherwise fails 340. The result eventually would be display and stored on a mobile device or transfer the data to a cloud 320.

[0217] In FIG. 7, the embodiment schematically illustrates the steps for nucleic acid amplification in point of collection at a higher level. The steps begins with collecting a sample from a test subject 360, performing nucleic acid amplification reaction 370, taking an image 380 and analyzing the image and finally determining if target nucleic acid is present in the test subject or not 400. The result will report to a cloud device 420. The analysis method used in 400 may include a statistics method. An exemplary statistics method is t-test or Anova.

[0218] Or it can also start with collecting samples 360 and performing nucleic acid amplification 390. The amplified nucleic acid is then sequenced 390. The sequencing data are analyzed and determine presence of target nucleic acid 410. The result will report to a cloud device 420.

[0219] In FIG. 8, the embodiment schematically illustrates the operation steps, procedures and reaction stages of an embodiment method. In terms of the operation stages, the method starts with sample collection 430, sample preparation 440, nucleic acid amplification for the sample 450, detect the nucleic acid amplification product 460, analyze the result to determine if the target nucleic acid is in the sample 470. In terms of procedures, it starts with collecting a sample 480, introducing the sample into a reaction chamber with cell lysis reagents, and then transfer the cell lysate into another reaction chamber 490, keep transferring the reaction chamber and cause it to contact the hot plate in the order of 95 deg C. 500, 55 deg C. 510, 72 deg C. 520, respectively, for a specific time, and repeat the cycle 35 times. Finally, the reaction chamber is moved to a suitable position, the image of nucleic acid amplification product is taken and analyzed 500. The result is 530 then reported to the user or cloud. In terms of reaction, it starts with cell lysis reaction 550 following by DNA denaturation 560, DNA annealing 570 and DNA synthesis 575.

[0220] In FIG. 9, the embodiment schematically illustrates a mobile device 580 is adapted with a nanopore sequencer 590, which may sequence the amplified nucleic acid prepared in a reaction chamber of the test platform.

[0221] In FIG. 10, the embodiment shows the histogram of hue values obtained from control and treatment samples. The hue values are obtained from the images of PCR products at the control and treatment group. The PCR is performed with the setup described in FIG. 1 and following by the steps-incubate the reaction chamber (a 0.2 ml PCR tube) at 90 deg. for 1 minute, and then start a PCR cycle-95 deg. 20 seconds C, 72 deg. C. 20 seconds, 43 deg. 20 seconds for 45 cycles. The DNA product is generated by amplifying the target nucleic acid 25 ng from M13 phage with 1 uM for both universal M13 forward and reverse primers, and Accuris™ Tag Plus PCR master mix. The 1×SYBR green dye is added for imaging. When imaging, an LED is under the reaction chamber (a 0.2 ml PCR tube) and emits 470 nm light. The control sample has no M13 phage DNA. The hue values are converted from RGB values of images from the treatment and control samples, respectively. The hue values determined herein are: 182, 179, 182, 181, 177 for samples from treatment group (with 25 ng M13 phage DNA) while 224, 227, 228, 228, 228 are for control samples or samples from control group. The p value for one tail t-test is 2.85E-06.

[0222] In FIG. 11, the exemplary embodiment shows one electric thermos 680 filled with water and has a predesigned temperature as a heat source. A test platform 600 is supported by the thermos. On the platform, there is a step motor 610 connected to an arm 620. The arm hangs up a receptacle 630. And the receptacle accommodates a tube 640 for a sample. The motor is powered by a battery 650. The position and duration of the arm is controlled by a programmed Arduino UNO R3 board 660 and ULN2003 control board 670. The other tubes 710 contains a control sample and can serve as a color calibration. The temperature label 700 may be used for monitor the temperature of reaction with colorimetric method. By shuttling the arm, the tube may immerse into the thermos—the proximity of a heat source—for a particular period of time. Since the thermo may have a temperature corresponding to isothermal amplification reaction, immersing the tubes into the thermos may change the temperature of nucleic acid amplification reaction, and cause the reaction to complete. Once the reaction is complete, the step motor may drive the arm and move the receptacle out of thermo to a position—a measurement position—that is not over the thermo and suitable a user to take the amplified nucleic acid for nanopore sequencing. Or the measurement position is suitable for a user to measure the color change of product due to the amplified nucleic acid via the camera of a mobile device 690.

[0223] In FIG. 12, a tourbillon is used to shuttle the reaction chambers in a test platform between different heat sources. A clock hand of a tourbillion can serve as an arm 710 and circle around three chemically activated heat sources 770 at three constant temperatures, respectively (as the chemically activated heat source illustrated in FIG. 4). The arm may hang up a receptacle 720 for hosting a reaction chamber 730. The reaction chamber may have a polymerase chain reaction. The reaction chamber may have a thermal contact with the heat sources when the reaction chamber immersed into the water of a heat source. The arm shuttles the reaction chamber to each heat source for each stage of PCR-95 deg. C. for DNA denaturation, 55 deg. C. for primer annealing, 72 deg. C. for DNA synthesis. Once the arm finishes a round, the PCR reaction in the reaction chamber also finish a cycle. The arm can be power by the spring of tourbillion 740 and drives the reaction chamber to the front of a camera of a mobile device 750 by a gear set 780. A LED 760 might be optional for using the colorimetric method to determine amplification result. Thereby, the usage of electricity for nucleic acid amplification in the current disclosure is optional.

EXEMPLIFICATIONS

[0224] Example 1: In this example, as configured in FIG. 1, the system comprises three electric thermos filled with water with the temperatures 95 deg. C., 72 deg. C., 55 deg. C. respectively. One may prepare a DNA sample from a saliva sample (treatment group) by mixing a lysis buffer (i.e. 50 unit/ml Proteinase K with TE buffer) and the saliva in a PCR tube on a floating rack. The tube with the floating rack may be put into the 55 deg. C. thermo for 15 minutes (or follow the method described in Genome Res. 4: 368-370 (1995)) and then 95 deg. C. thermo for 5 minutes as taught in a reference (Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP, medRxiv. Preprint. 2020 May 11). After the nucleic acid extraction step, the cell lysate as well as PCR master mix and a primer set is then introduced into a PCR tube on the receptacle of a test platform. The test platform sits over three thermos and has an arm driven by a step motor. The arm hangs up the receptacle of tubes (or reaction chambers). By rotating the arm, the tubes (or reaction chambers) may immerse into water in different thermos at each time when the arm moves right above the thermo. Thereby, via rotating the arm, the temperature of reaction in PCR tubes may be controlled. Also, by changing the duration of holding the arm over a particular thermos, the reaction time can be controlled as well. Thereby, by rotating the arm over the thermos with the order: above 95 deg. C. thermo for 15 seconds, 55 deg. C. thermos for 15 second and 55 deg. C. thermos for 15 second, one PCR cycle may be complete. By repeating the same sequence and move the receptacle over the thermos, the nucleic acid amplification reaction may produce enough DNA for colorimetric measurement or DNA sequencing. Once a desired number of PCR cycles is reached, the arm may rotate to a position that is above a cell phone camera. One may add SYBR Green dye into the reaction, and turn on a 395 nm LED light beneath the tube. An image for both the tube and color calibration is taken by the camera on a cell phone. The control sample (control group, which may serve as a color calibration as well) is another tube with the PCR reagents, primers set and SYBR Green dye but DNA from saliva sample (treatment group). The image is processed by retrieving the RGB values from both tubes from the treatment and control group (or a color calibration). The RGB values are then converted to hue values. If the difference of hue values between the saliva sample (treatment group) and control sample (control group) is above a threshold, the target DNA may present in the saliva sample.

[0225] The DNA produced in this way can be collect and preserve for a portable nucleic acid sequencer such as a nanopore sequencer. Following the procedure instructed in (Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples, Nature Protocols, 12, 1261 (2017)), one may sequence the amplified DNA and processed the sequencing data from a mobile device. Thereby, a genome sequencing information may be obtained in a point-of-collection manner. Since the thermo with a temperature control can be easily obtained at a low cost, and the thermo may be used for drink or other beverage after all. The current disclosure is particularly suitable for location with a very limited resource. Furthermore, in term of period of duration for completing one PCR cycle, the current disclosure requires less a than half of time than a convention thermal cycler for a 100 bp DNA synthesis, which requires heating up or cooling down a heating block before reaching a predesigned temperature.

[0226] Example 2: In one embodiment, a carrier may have four wheels and is able to move linearly. A receptacle may sit in the carrier. The receptacle is able to accommodate 1600 reaction chambers and has an area of 60 cm×60 cm. In one embodiment, the samples may be collected by capillaries shown in FIG. 5. and introduced into the receptacle. In one embodiment, the receptacle may have pre-dry primer set and/or wax beads with PCR master mix before the samples are introduced. In one embodiment, the primer sets may cover different genome locations of one or more organisms. In one embodiment, the receptacle may also be able to contact with the top surface of a silicon rubber heater. There may be three silicon rubber heaters, and each may have a top surface area 70 cm×70 cm with preset temperature 95 deg. C., 72 deg. C., 55 deg. C. respectively. These silicon rubber heaters may be aligned in a line so that the carrier may move over them in a direction. The carried is driven by motors and may translocate the receptacle over each silicon rubber heater for each predetermined time, and complete the nucleic acid amplification reaction after a certain number of PCR cycles. Thereby, the 1600 nucleic acid amplification reactions can all be complete at once. Since a silicon rubber heater is easy to pack and carry, and a mobile device is easy to access, the current disclosure is particularly useful in certain locations. Such as a farm or a remote area, sometime, a large number of nucleic acid reactions needs to be carried out but a high throughput facility is not available.

[0227] Example 3: In one embodiment, plurality of reaction chambers may contain identical primer sets. Thereby, plurality of identical reactions may be carried out under the same conditions. If there are three or more samples collected from each a control group and a treatment group, respectively, a proper statistics method such as t-test or Analysis of variance (ANOVA) can be used to determine the confidence level of results. Since each hue value can be obtained from the colorimetric measurements of each reaction, the hue values may be used to determine if a null hypothesis—the samples from control group are identical to the samples from treatment group—is valid under certain confidence level such as p value below 0.05.

[0228] Example 4: In one embodiment, a temperature label can associate with a heat source or a reactive test region, and the temperature label changes color when the temperature of heat source or reactive test region changes. Thereby, the temperature of a heat source or reactive test region is monitored via images taken by a mobile device. The mobile device can be installed with software. The software can process the images for the color coordinates and determine the temperature of the heat source or reactive test region.

[0229] Example 5: In one embodiment as shown in FIG. 12, a tourbillon is used to shuttle the reaction chambers in a test platform between different heat sources. A clock hand of a tourbillion can serve as an arm and circle around three chemically activated heat sources at three different temperatures (as the chemically activated heat source illustrated in FIG. 4). The arm may hang up a receptacle for hosting a reaction chamber. The reaction chamber may have a polymerase chain reaction. The reaction chamber may have a thermal contact with each heat source at once when the arm shuttles the reaction chamber for each different amplification stage. Each time, when the arm of tourbillion completes a round, one PCR cycle can be complete as well. Thereby, the usage of electricity in current disclosure is optional.

[0230] While the present invention has been described above in terms of specific embodiments, it is to be understood that the invention is not limited to these disclosed embodiments. Many modifications and other embodiments of the invention will come to mind of those skilled in the art to which this invention pertains, and which are intended to be and are covered by both this disclosure and the appended claims. It is indeed intended that the scope of the invention should be determined by proper interpretation and construction of the appended claims and their legal equivalents, as understood by those of skill in the art relying upon the disclosure in this specification and the attached drawings.