A high-throughput detection method based on RPA-CRISPRCas12a platform and microfluidic chip

Abstract

The present invention discloses a microfluidic chip and a high-throughput detection method based on the RPA-CRISPRCas12a platform and the microfluidic chip. The method comprises the following steps: S1: providing a microfluidic chip, loading RPA and CRISPR / Cas12a reaction systems into the first and second reaction chambers respectively; S2: injecting the sample to be tested from the sample injection hole, pushing the push rod to allow the liquid to enter the lower structure until it enters the first reaction chamber, and performing an RPA amplification reaction; S3: flipping the microfluidic chip and pushing the push rod to allow the RPA product to enter the second reaction chamber, and performing a CRISPR / Cas12a reaction; S4: using a portable ultraviolet flashlight and a filter to analyze the results. The present invention constructs a new method for simultaneously detecting four pathogenic microorganisms. The sample contains four targets, and the detection results do not have cross-interference. It has good stability, accuracy, repeatability and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid molecule target detection, and more specifically relates to a high-throughput detection method based on an RPA-CRISPR Cas12a platform and a microfluidic chip. Background Art

[0002] Agricultural biosafety factors have become a major concern and a global health issue. With the rapid development of the agricultural product industry, timely testing for biosafety factors in agricultural products during production is essential. Traditional detection methods no longer meet the market demand for high-throughput, high-speed, and efficient testing, nor are they suitable for rapid on-site testing. Therefore, there is an urgent need to develop novel, rapid, and versatile point-of-care (POCT) diagnostic technologies with high sensitivity and specificity for quality control of agricultural biosafety factors throughout the agricultural industry chain, as well as for monitoring cleaning and hygiene practices.

[0003] In recent years, CRISPR / Cas endonuclease detection systems have shown great promise in molecular diagnostics due to their high sensitivity, specificity, and low cost. Currently, a large number of CRISPR / Cas systems combined with nucleic acid amplification technologies have been developed for rapid detection of safety factors, showing great potential for application in point-of-care (POCT) applications. This study established a rapid, efficient, and portable technology system and detection method based on isothermal nucleic acid target amplification technology and the precise recognition system of clustered regularly interspaced short palindromic repeats (CRISPR) Cas12a, combined with a high-throughput microfluidic chip. Summary of the Invention

[0004] The purpose of the present invention is to provide a microfluidic chip and a high-throughput detection method based on the RPA-CRISPRCas12a platform and the microfluidic chip, thereby solving the problem that the prior art urgently needs a new, rapid and multifunctional on-site detection and diagnosis method with high sensitivity and high specificity.

[0005] In order to solve the above problems, the present invention adopts the following technical solutions:

[0006] According to a first aspect of the present invention, there is provided a microfluidic chip, comprising: an upper structure, a lower structure, a push rod, a first reaction chamber, and a second reaction chamber; wherein the upper structure has a rectangular parallelepiped structure, comprising: a push rod accommodating cavity extending from the rear end toward the front end, a sample addition hole extending vertically through the front end of the push rod accommodating cavity, and four upper outlets arranged on the front end surface; the lower structure has a rectangular parallelepiped structure, comprising: a flow channel extending on its top surface, the top surface cooperates with the bottom surface of the upper structure, the flow channel comprises a main flow channel and four branch flow channels formed by two diversions, the inlet position of the main flow channel is the same as the inlet position of the lower structure. The bottoms of the sample loading holes in the upper structure are aligned in the vertical direction, and the ends of the four branch flow channels are provided with lower outlets extending through in the vertical direction; the first reaction chamber is composed of four first PCR reaction tubes connected in a row, and these four first PCR reaction tubes are respectively connected to the four lower outlets at the bottom of the lower structure; and the second reaction chamber is composed of four second PCR reaction tubes connected in a row, and these four second PCR reaction tubes are respectively connected to the four upper outlets at the front end of the upper structure; wherein, by flipping the microfluidic chip upside down and pushing the push rod, the flow of liquid from the first reaction chamber to the second reaction chamber can be achieved.

[0007] Preferably, the upper structure and the lower structure are bonded together by glue.

[0008] Preferably, the bottom of the upper structure is provided with four grooves respectively aligned with the four lower-layer outlets in the lower structure, and the four grooves are respectively connected to the four upper-layer outlets through an upper-layer flow channel.

[0009] Preferably, the push rod is interference-fitted with the inner wall of the push rod accommodating cavity, and liquid pushing can be achieved by pushing the push rod under the action of external force.

[0010] Preferably, the upper structure and the lower structure are made of PMMA material.

[0011] Preferably, the four upper outlets at the front end of the upper structure and the four lower outlets at the bottom of the lower structure are arranged in a straight line.

[0012] According to a second aspect of the present invention, a high-throughput detection method based on an RPA-CRISPRCas12a platform and a microfluidic chip is provided, comprising the following steps: S1: providing a microfluidic chip as described in any one of claims 1 to 6, loading an RPA reaction system into a first reaction chamber, and loading a CRISPR / Cas12a reaction system into a second reaction chamber; S2: after the sample to be tested and MgOAC are mixed evenly, the sample is injected from the sample injection hole of the upper structure, and the push rod is pushed to allow the liquid to enter the flow channel of the lower structure until it enters the first reaction chamber, and then the first reaction chamber is placed in a metal bath for incubation at 38-42°C for 8-12 minutes to perform an RPA amplification reaction; S3: the microfluidic chip is turned over so that the liquid in the first reaction chamber falls into the groove at the bottom of the upper structure, the push rod is pushed to allow the RPA product to enter the second reaction chamber along the upper flow channel, and then the second reaction chamber is placed in a metal bath for incubation at 38-42°C for 8-12 minutes to perform a CRISPR / Cas12a reaction; S4: the results are analyzed using a portable UV flashlight and a filter.

[0013] RPA primers designed for one detection target are added to each of the four first PCR reaction tubes in the first reaction chamber, and crRNA designed for one detection target is added to each of the four second PCR reaction tubes in the second reaction chamber, thereby ultimately achieving simultaneous detection of four detection targets.

[0014] When the high-throughput detection method is used for the simultaneous detection of hly, tlh, nuc and rfbE genes, the RPA primer sequences designed for hly, tlh, nuc and rfbE genes are shown in SEQ ID NO.1-2, SEQ ID NO.4-5, SEQ ID NO.7-8, and SEQ ID NO.10-11, respectively, and the crRNA sequences designed for hly, tlh, nuc and rfbE genes are shown in SEQ ID NO.3, 6, 9, and 12, respectively.

[0015] In step S3, when 6 μL of the RPA product was added to the second reaction chamber, the detection sensitivity was the highest.

[0016] It should be noted that in a study proposed by Wu et al. on a high-throughput microfluidic strategy for detecting Listeria monocytogenes based on RAA-CRISPR / Cas13a dual signal amplification, the reagents needed to be added to the reaction chamber by adding the sample port and then placed in a rotator, followed by freeze-drying. This involved cumbersome steps and a complex method. However, the present invention only requires installing or replacing a reaction chamber composed of PCR tubes to add the reagents to the reaction system, eliminating the need for pre-embedding, freeze-drying, and other operations. The chip designed by the present invention does not require pre-embedding of reagents, and both reaction chambers can be easily disassembled for easy operation.

[0017] It is also known that a microfluidic detection method based on capillary force is disclosed in the prior art. However, this type of non-self-driven chip needs to be connected to a pump to achieve the purpose of allowing the liquid to flow within the chip. However, these necessary devices are not conducive to on-site detection. The present invention designs such a self-driven multiple detection microfluidic chip, which uses the dead weight of the liquid to achieve liquid drive and complete detection during detection. Therefore, compared with the prior art, the present invention realizes self-drive, eliminates external power devices such as pumps, and does not need to be connected to detection instruments such as mass spectrometers, while being faster and more time-saving.

[0018] In summary, according to a microfluidic chip provided by the present invention and a high-throughput detection method based on the RPA-CRISPRCas12a platform and microfluidic chip, a new method (RPA-CRISPR / Cas12a-SDMC) for the simultaneous detection of four pathogenic microorganisms is constructed. Even if the sample contains four targets at the same time, there is no cross-interference in the test results, and it has good stability, accuracy and repeatability. At the same time, the method can reach 10CFU / mL for the LOD of pure bacterial culture. The detection sensitivity for rfbE gene and tlh gene reaches 10fg / μL, and the detection sensitivity for hly gene and nuc gene is 100fg / μL, so the detection method has high sensitivity. The whole detection process is completed within 40min. The microfluidic chip designed by the present invention is a self-driven chip, which does not need to be connected to instruments such as pumps and the results do not require instrumental analysis, so the method meets POCT. In addition, the method is time-saving and simple to operate than the single detection method. It has broad application prospects in the field of nucleic acid molecule target detection technology, simplifies food detection supervision process, and saves manpower and material resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the structure of a microfluidic chip provided according to a preferred embodiment of the present invention; (A) a top view of the upper and lower structures of the microfluidic chip; (B) a side view of the microfluidic chip; (C) a schematic diagram of the assembly of the overall structure of the microfluidic chip;

[0020] Figure 2 The operational process of the RPA-CRISPR / Cas12a-SDMC platform is shown; (A) adding the test sample; (B) RPA amplification reaction; (C) adding the RPA product to the CRISPR / Cas12a system; (D) CRISPR / Cas12a enzyme cleavage reaction; (E) analyzing the results;

[0021] Figure 3Feasibility analysis of the RPA-CRISPR / Cas12a-SDMC platform is shown; (A) Sample contains one detectable target; (B) Sample contains two detectable targets; (C) Sample contains three detectable targets; (D) Sample contains four detectable targets; p < 0.05, three parallel experiments were tested;

[0022] Figure 4 The optimization of the amount of RPA reaction product added in the RPA-CRISPR / Cas12a-SDMC platform assay is shown; p < 0.05, three parallel experiments were tested, N: negative control, no template added;

[0023] Figure 5 Figure 2 shows the sensitivity analysis of the RPA-CRISPR / Cas12a-SDMC platform for detecting four genomes. (A) Analysis of the sensitivity of the RPA-CRISPR / Cas12a-SDMC platform for detecting four genomes. (B) Fluorescence values ​​of the RPA-CRISPR / Cas12a-SDMC platform detection results read by a multifunctional microplate reader. p < 0.05, tested in triplicate. N: Negative control, no template added.

[0024] Figure 6 Figure 2 shows sensitivity analysis of pure bacterial cultures detected by the RPA-CRISPR / Cas12a-SDMC platform. (A) Analysis of the sensitivity of the RPA-CRISPR / Cas12a-SDMC platform for four pathogenic bacteria. (B) Fluorescence values ​​of the RPA-CRISPR / Cas12a-SDMC platform assays read using a multifunctional microplate reader. p < 0.05, tested in triplicate. N: Negative control, no template added. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and materials used in the examples are commercially available unless otherwise specified.

[0026] Example 1 Construction of a microfluidic chip

[0027] According to this embodiment, a microfluidic chip is constructed using Designed. Figure 1 As shown, the microfluidic chip includes: an upper structure 10 , a lower structure 20 , a push rod 30 , a first reaction chamber 40 , and a second reaction chamber 50 .

[0028] The upper structure 10 and the lower structure 20 are both substantially rectangular. The upper structure 10 includes: a rear end ( Figure 1 Center left) toward the front ( Figure 1 The push rod accommodating chamber 11 extending from the center right side is used to accommodate the push rod 20. The sample loading hole 12 extends vertically through the front end of the push rod accommodating chamber 11, and four upper layer outlets 13 are provided on the front end surface. These four upper layer outlets 13 are arranged in a straight line. The lower structure 20 includes: a flow channel extending from the top surface, the top surface cooperates with the bottom surface of the upper structure, the flow channel includes a main channel 21 and four branch flow channels 22 formed by two diversions. The inlet position of the main channel 21 is vertically aligned with the bottom of the sample loading hole 12 in the upper structure. The ends of the four branch flow channels 22 are provided with lower layer outlets 23 extending vertically through the upper structure. These four lower layer outlets 23 are arranged in a straight line.

[0029] The first reaction chamber 40 is composed of four first PCR reaction tubes connected in a row. These four first PCR reaction tubes are connected to the four lower-level outlets 23 at the bottom of the lower structure 20. The second reaction chamber 50 is composed of four second PCR reaction tubes connected in a row. These four second PCR reaction tubes are connected to the four upper-level outlets 13 at the front end of the upper structure 10.

[0030] The upper structure 10 and the lower structure 20 are optionally bonded together using glue, so that the bottom surface of the upper structure 10 mates with the top surface of the lower structure 20. The push rod 30 forms an interference fit with the inner wall of the push rod accommodating cavity 11, so that pushing the push rod 30 under external force can achieve liquid push. By flipping the microfluidic chip upside down and pushing the push rod 30, liquid can flow from the first reaction chamber 40 to the second reaction chamber 50.

[0031] According to this preferred embodiment, the bottom of the upper structure 10 is further provided with four grooves 14, which are aligned with the four lower-layer outlets 23 in the lower structure 20. These four grooves 14 are connected to the four upper-layer outlets 13 via an upper-layer flow channel 15. Therefore, after the RPA amplification reaction is completed, the microfluidic chip is flipped over, and the RPA product in the first reaction chamber 40 flows into the grooves 14 under the action of gravity. At this time, the push rod 30 is pushed again, and the RPA product flows through the upper-layer flow channel 15 until it reaches the second reaction chamber 50.

[0032] According to the preferred embodiment, the upper structure and the lower structure are made of polymethyl methacrylate (PMMA) plates.

[0033] According to this preferred embodiment, the total length, width, and height of the microfluidic chip are 67 mm, 35 mm, and 16.47 mm, respectively. The lower structure 20 is slightly shorter than the upper structure 10. In the upper structure 10, the plunger cavity 11 has a diameter of 8.7 mm and a length of 19 mm, which can accommodate the plunger of a 1.5 mL syringe. The sample injection port 12 has a depth of 12 mm. The total length of the flow channel extending from the top surface of the lower structure 20 is approximately 28 mm, and the maximum width of the flow channel is 0.3 mm. The groove 14 at the bottom of the upper structure 10 is approximately 3.36 mm deep. When the chip is inverted, the liquid in the reaction tube flows into the groove 14 under the action of gravity. It should be understood that the amount of RPA product added to the second reaction chamber is determined by the volume of the groove 14. The length of the upper flow channel 15 connecting the groove 14 to the upper outlet 13 is 10 mm. However, it should be understood that this is merely a preferred embodiment and not a limitation, and the microfluidic chip provided by the present invention is not limited to the above dimensions.

[0034] Example 2 provides a high-throughput detection method based on RPA-CRISPRCas12a platform and microfluidic chip

[0035] 2.1 Primer and crRNA design for RPA and CRISPR / Cas12a

[0036] According to this embodiment, a high-throughput detection method based on the RPA-CRISPRCas12a platform and a microfluidic chip is provided, and is used for simultaneous detection of hly (from Listeria monocytogenes, gene accession number MG922920.1), tlh (from Vibrio parahaemolyticus, gene accession number M36437), nuc (from Staphylococcus aureus, gene accession number DQ507382.1), and rfbE (from Escherichia coli O157:H7, gene accession number S83460) genes. The RPA primers designed for the hly, tlh, nuc and rfbE genes, and the crRNA sequences are shown in Table 1 below, respectively.

[0037] Table 1 RPA primers and crRNA sequences of four bacteria (SEQ ID NO.1-12)

[0038]

[0039]

[0040] 2.2RPA and CRISPR / Cas12a reaction system

[0041] The RPA procedure was performed according to the RPA assay kit instructions. First, thoroughly mix 29.5 μL of Primer Free Rehydration Buffer with 8.2 μL of ddH2O and 2.4 μL of primers. This was then added to the dry enzyme powder, completely dissolved, and stored at -20°C until ready for use. DNA and MgOAC were added last. The reaction was incubated at 40°C for 10 minutes.

[0042] The CRISPR / Cas12a reaction system consisted of 50 nM LbCpf1 nuclease, 10 μL RNA inhibitor, 500 nM crRNA, 500 nM ssDNA, 2 μL NE buffer, and 6 μL RPA reaction product in a 20 μL reaction volume. The reaction was incubated at 40°C for 10 min.

[0043] 2.3 Detection process of RPA-CRISPR / Cas12a-SDMC platform

[0044] The first reaction chamber 40 equipped with the RPA reaction system and the second reaction chamber 50 equipped with the CRISPR / Cas12a reaction system were respectively installed on the microfluidic chip. First, 20 μL of the sample to be tested and 10 μL of MgOAC were mixed evenly and then added to the microfluidic chip from the sample addition hole 12 (as shown in FIG Figure 2 Then, seal the sample loading hole 12 with tape and push the push rod 30 to allow the sample to enter the flow channel extending from the top surface of the lower structure 20. Then, mix the liquid in the reaction tube 1 evenly and place it in a portable metal bath at 40°C for 10 minutes (as shown in FIG. Figure 2 After the RPA amplification reaction, the chip is flipped over so that the liquid in the first reaction chamber 40 falls into the groove 14 at the bottom of the upper structure (as shown in FIG. Figure 2 Then push the push rod 30 to allow the reaction product to enter the second reaction chamber 50 (as shown in C). Figure 2 The liquid was mixed evenly and placed in a portable metal bath and incubated at 40°C for 10 minutes. Figure 2 As shown in Figure E, the results were analyzed using a portable UV flashlight and filters.

[0045] Example 3 Feasibility analysis of high-throughput detection on the RPA-CRISPR / Cas12a-SDMC platform

[0046] In order to analyze the feasibility of the RPA-CRISPR / Cas12a-SDMC platform, this example prepared samples containing one, two, three, and four targets (i.e., hly, tlh, nuc, and rfbE genes), a total of 15 combinations, and tested them using the RPA-CRISPR / Cas12a-SDMC platform. The test results are shown in Figure 2. Figure 3As shown, no matter how many detection targets are contained in the sample, the corresponding targets can be detected by the method of the present invention, and no fluorescence is generated in the test tubes of other targets, that is, no cross contamination occurs. Therefore, this method is not only feasible, but also highly specific, and realizes high-throughput detection.

[0047] Example 4 Optimization of the amount of RPA reaction product added in RPA-CRISPR / Cas12a-SDMC platform detection

[0048] In order to make the detection results more convenient for visual inspection and improve the detection sensitivity, the amount of RPA product added was optimized in this embodiment. Figure 4 As shown in the figure, when the amount of RPA product added was 6 μL, the fluorescence values ​​of the four groups were all higher than those of other addition amounts. Therefore, the amount of RPA product added in subsequent experiments was preferably 6 μL.

[0049] Example 5 Genomic Sensitivity Analysis of RPA-CRISPR / Cas12a-SDMC Platform Detection

[0050] In order to evaluate the sensitivity of the platform to the genome, this example prepared 6 ~10 0 fg / μL of the specific pathogenicity genes (rfbE, nuc, tlh and hly) of four pathogens. Figure 5 As shown in Figure A, the detection sensitivity of the RPA-CRISPR / Cas12a-SDMC platform for the rfbE gene in Escherichia coli O157:H7 and the tlh gene in Vibrio parahaemolyticus was 10 fg / μL, which was consistent with the fluorescence intensity results (Figure 2A). Figure 5 The detection sensitivity of the RPA-CRISPR / Cas12a-SDMC platform for the nuc gene in Staphylococcus aureus and the hly gene in Listeria monocytogenes was 100 fg / μL, which was consistent with the fluorescence intensity results (as shown in Figure 2). Figure 5 B2 and B4 in FIG.

[0051] Example 6 Genomic Sensitivity Analysis of RPA-CRISPR / Cas12a-SDMC Platform Detection

[0052] In order to evaluate the sensitivity of the platform to pure bacterial culture, this example prepared 6 ~10 0 CFU / mL of pure culture mixture of four pathogenic bacteria. Figure 6 As shown in Figure C, the LOD of the RPA-CRISPR / Cas12a-SDMC platform for pure culture of four bacteria was 10 CFU / mL, which is consistent with the fluorescence intensity results (as shown in Figure 4). Figure 6This demonstrates that the RPA-CRISPR / Cas12a-SDMC platform provided by the present invention can simultaneously detect four types of bacteria with relatively high sensitivity.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. Any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention are within the scope of protection of the patent claims. Anything not fully described in this invention is conventional technology.

Claims

1. A microfluidic chip, characterized in that: include: Upper structure, lower structure, push rod, first reaction chamber, second reaction chamber; wherein, The upper structure has a rectangular parallelepiped structure, including: a push rod accommodating cavity extending from the rear end toward the front end, a sample addition hole extending vertically through the front end of the push rod accommodating cavity, and four upper layer outlets arranged on the front end surface; The lower structure has a rectangular parallelepiped structure, including: a flow channel extending from its top surface, the top surface being matched with the bottom surface of the upper structure, the flow channel including a main channel and four branch channels formed by two diversions, the inlet position of the main channel being vertically aligned with the bottom of the sample loading well in the upper structure, and the ends of the four branch channels being provided with lower layer outlets extending vertically through the channel; The first reaction chamber is composed of four first PCR reaction tubes connected in a row, and the four first PCR reaction tubes are respectively connected to the four lower layer outlets at the bottom of the lower structure; and The second reaction chamber is composed of four second PCR reaction tubes connected in a row, and the four second PCR reaction tubes are respectively connected to the four upper outlets at the front end of the upper structure; The flow of liquid from the first reaction chamber to the second reaction chamber can be achieved by turning the microfluidic chip upside down and pushing the push rod.

2. The microfluidic chip according to claim 1, characterized in that The upper structure and the lower structure are bonded together by glue.

3. The microfluidic chip according to claim 1, characterized in that The bottom of the upper structure is provided with four grooves which are aligned with the four lower layer outlets in the lower structure respectively. The four grooves are connected with the four upper layer outlets respectively through an upper layer flow channel.

4. The microfluidic chip according to claim 1, characterized in that The push rod is interference-fitted with the inner wall of the push rod accommodating cavity, and liquid pushing can be achieved by pushing the push rod under the action of external force.

5. The microfluidic chip according to claim 1, characterized in that The upper structure and the lower structure are made of PMMA material.

6. The microfluidic chip according to claim 1, characterized in that The four upper outlets at the front end of the upper structure and the four lower outlets at the bottom of the lower structure are arranged in a straight line.

7. A high-throughput detection method based on the RPA-CRISPRCas12a platform and microfluidic chip, characterized in that: The following steps are involved: S1: Provide a microfluidic chip according to any one of claims 1 to 6, wherein the first reaction chamber is loaded with an RPA reaction system, and the second reaction chamber is loaded with a CRISPR / Cas12a reaction system; S2: After the sample to be tested and MgOAC are evenly mixed, the sample is injected from the sample injection hole of the upper structure, and the push rod is pushed to allow the liquid to enter the flow channel of the lower structure until it enters the first reaction chamber. The first reaction chamber is then placed in a metal bath and incubated at 38-42°C for 8-12 minutes to perform RPA amplification reaction; S3: Flip the microfluidic chip so that the liquid in the first reaction chamber falls into the groove at the bottom of the upper structure, push the push rod to allow the RPA product to enter the second reaction chamber along the upper flow channel, and then place the second reaction chamber in a metal bath and incubate at 38-42°C for 8-12 minutes to perform the CRISPR / Cas12a reaction; S4: Analyze the results using a portable UV flashlight and filters.

8. The high-throughput detection method according to claim 7, characterized in that RPA primers designed for one detection target are added to each of the four first PCR reaction tubes in the first reaction chamber, and crRNA designed for one detection target is added to each of the four second PCR reaction tubes in the second reaction chamber, thereby ultimately achieving simultaneous detection of four detection targets.

9. The high-throughput detection method according to claim 7, characterized in that When the high-throughput detection method is used for the simultaneous detection of hly, tlh, nuc and rfbE genes, the RPA primer sequences designed for hly, tlh, nuc and rfbE genes are shown in SEQ ID NO.1-2, SEQ ID NO.4-5, SEQ ID NO.7-8, and SEQ ID NO.10-11, respectively, and the crRNA sequences designed for hly, tlh, nuc and rfbE genes are shown in SEQ ID NO.3, 6, 9, and 12, respectively.

10. The high-throughput detection method according to claim 7, characterized in that In step S3, when 6 μL of the RPA product was added to the second reaction chamber, the detection sensitivity was the highest.

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