Rapid detection method for fusarium wilt in corn based on RPA-CRISPR / Cas12b technology

Abstract

The invention discloses a rapid detection method for fusarium wilt in corn based on an RPA-CRISPR / Cas12b technology, and belongs to the technical field of nucleic acid detection. The invention provides a specific reagent combination, a kit and a detection method in order to solve the problems of high amplicon pollution risk, complicated operation and the like in the existing technologies such as RPA (recombinase polymerase amplification). The reagent combination comprises an RPA (recombinase polymerase amplification) primer pair with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, and sgRNA (single guide ribonucleic acid) with a sequence as shown in SEQ ID NO.3. The method comprises the following steps: carrying out RPA amplification by taking nucleic acid of a sample to be detected as a template, carrying out mixed incubation on an amplification product, sgRNA, Cas12b protein and fluorescent reporter molecules, and judging a result by detecting a fluorescent signal. The optimized RPA and CRISPR systems are integrated on the centrifugal micro-fluidic chip, so that the full-flow closed tube and automatic detection is realized, the detection can be completed within 30 minutes, the detection sensitivity reaches 1E2 copies / T, the risk of aerosol pollution is greatly reduced, and the centrifugal micro-fluidic chip is suitable for port quarantine and field on-site rapid detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nucleic acid detection, and particularly relates to a rapid detection method for corn inner state wilt fungus based on RPA-CRISPR / Cas12b technology. BACKGROUND

[0002] Point-of-Care Testing (POCT) of plant pathogenic microorganisms is the key to protect crop production safety and carry out effective quarantine. Corn inner state wilt fungus (Gossyphium subsp. Clavibacter michiganensis subsp. nebraskense , CMN) as an important quarantine bacteria can cause serious loss of corn yield, and its rapid detection is of great significance for prevention and control.

[0003] At present, the detection of CMN mainly relies on molecular detection techniques such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). PCR technology has high sensitivity and good specificity, but it is seriously dependent on a thermal cycler, and the operation is complicated and time-consuming, which is difficult to apply to on-site rapid detection. The isothermal amplification technologies such as LAMP and RPA overcome the dependence on temperature cycle to a certain extent, significantly shorten the amplification time, and provide the possibility for the development of on-site detection.

[0004] However, these methods still have obvious limitations: first, most of the detection still needs to be opened after completion for electrophoresis or test strip detection, which increases the risk of aerosol pollution and easily leads to false positive results; second, the whole process involves multiple steps of operation, which is difficult to integrate, limiting its application effect in the field environment such as ports and fields.

[0005] Therefore, there is an urgent need in the art to develop a detection method that can integrate nucleic acid rapid amplification, specific detection and closed tube operation into one, to realize reliable detection of CMN on-site, rapidly, high sensitivity and anti-pollution. SUMMARY

[0006] In order to solve the problems of high amplicon pollution risk, complicated operation and dependence on large equipment in the existing nucleic acid detection technology of plant pathogenic microorganisms, especially the RPA technology in on-site rapid detection, the application provides a rapid detection method for corn inner state wilt fungus based on RPA-CRISPR / Cas12b technology. The method realizes the full-process automation, closed tube, high sensitivity, high specificity and rapid detection from sample loading to result interpretation by designing specific RPA primers and sgRNA, optimizing isothermal amplification system and CRISPR detection system, and integrating into a centrifugal microfluidic chip, thereby eliminating the amplicon pollution risk and significantly improving the detection efficiency and on-site applicability.

[0007] To achieve the above-mentioned purposes of the application, the following technical solutions are adopted in the application: The application provides a reagent combination for detecting Clavibacter michiganensis subsp. nebraskense, comprising a specific RPA primer pair and sgRNA. The RPA primer pair is composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2; the sgRNA can target a specific sequence in the product amplified by the RPA primer pair, and has a nucleotide sequence as shown in SEQ ID NO. 3.

[0008] The application further provides a kit for detecting Clavibacter michiganensis subsp. nebraskense, comprising: (1) the reagent combination; (2) Cas12b protein; (3) ssDNA reporter molecule, which is labeled with a fluorescent reporter group at one end and a quenching group at the other end.

[0009] The application further provides a method for detecting Clavibacter michiganensis subsp. nebraskense for non-diagnostic purposes, comprising the following steps: S1, using the nucleic acid of a sample to be tested as a template, performing RPA reaction using a specific primer pair with nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2; S2, CRISPR detection reaction: mixing the amplification product with the sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule and performing constant temperature incubation; S3, detecting the fluorescent signal, and determining whether the sample to be tested contains Clavibacter michiganensis subsp. nebraskense according to the fluorescent signal.

[0010] Further, the RPA reaction is performed at 43℃ for 15 minutes; and the constant temperature incubation is performed at 43℃ for 10 minutes.

[0011] Further, the RPA reaction, CRISPR detection reaction and fluorescent signal detection are all automatically completed on a centrifugal microfluidic chip; the centrifugal microfluidic chip comprises an RPA reaction cavity and a CRISPR detection cavity which are mutually isolated and in which the RPA primer pair and sgRNA in the reagent combination are pre-disposed. Advantages

[0012] (1) The present application has high sensitivity and specificity for the detection of corn northern wilt pathogen (CMN) by combining the specific RPA primer (SEQ ID NO. 1 and SEQ ID NO. 2) and sgRNA (SEQ ID NO. 3) designed with care, and using the double recognition and signal amplification of the target sequence of the CRISPR-Cas12b system, so that the detection method has high sensitivity and specificity for the detection of corn northern wilt pathogen (CMN), and the minimum detection limit can reach 1E2 copies / T, which is much better than the conventional PCR method.

[0013] (2) The entire detection process of the present application, including RPA constant temperature amplification (43℃, 15min) and CRISPR detection reaction (43℃, 10min), can be completed within 30 minutes, greatly shortening the detection time and meeting the needs of on-site rapid detection (POCT).

[0014] (3) The present application integrates RPA amplification and CRISPR detection into a closed centrifugal microfluidic chip, realizes the whole process closed tube operation of "sample in, result out", avoids the aerosol pollution risk caused by opening the cover to detect the product in the conventional method, and significantly improves the reliability and accuracy of the detection result.

[0015] (4) The present application simplifies the complex multi-step operation into one-time sample addition by pre-freezing reagents and chip design. The detection process is automatically completed by a portable device, without the need for complex thermal cycler and professional operators, greatly reducing the use threshold, and is suitable for limited resources such as grassroots units, ports and field sites. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The present application is a three-dimensional structure schematic diagram of the centrifugal microfluidic detection platform used in the embodiment of the present application.

[0017] Figure 2 The present application is a structure decomposition and chamber layout schematic diagram of the microfluidic chip, mainly showing the sample bin, RPA reaction cavity, CRISPR detection cavity and connecting flow channel.

[0018] Figure 3 The present application is a flow process schematic diagram of the microfluidic chip in the working process, in which the reaction liquid flows through different chambers in a preset time sequence under the driving of centrifugal force.

[0019] Figure 4 The present application is a performance screening result diagram of three candidate sgRNAs of CMN target in the embodiment of the present application; (A) is the fluorescence change rate; (B) is the fluorescence curve.

[0020] Figure 5Figure for performance screening results of different RPA primer combinations of CMN target in the embodiments of the application; (A) is the fluorescence change rate; (B) is the fluorescence curve of each primer combination under high concentration template (1E4 copies / T); (C) is the fluorescence curve of each primer combination under low concentration template (1E3 copies / T).

[0021] Figure 6 Figure for sensitivity verification results of the detection method described in the application; (A) is the fluorescence change rate; (B) is the fluorescence curve.

[0022] Figure 7 Figure for sensitivity confirmation results of the detection method described in the application near the detection limit; (A) is the fluorescence change rate of 10 repeated detections; (B) is the fluorescence curve of high concentration template (5E2 copies / T); (C) is the fluorescence curve of low concentration template (1E2 copies / T).

[0023] Figure 8 Figure for detection effect of 1E4 copies / T target nucleic acid by the CRISPR detection system after dry treatment on the microfluidic chip.

[0024] Figure 9 Figure for whole-process closed tube detection effect of 1E4 copies / T target nucleic acid by the RPA primer and the CRISPR detection system after dry treatment on the microfluidic chip. DETAILED DESCRIPTION

[0025] In order to enable the personnel in the technical field to better understand the technical solutions in the application, the following will further describe the application with reference to the embodiments.

[0026] The nucleotide sequence involved in the application is as follows: SEQ ID NO. 1: the sequence shown is the nucleotide sequence of RPA primer CMN-RPA-F1.

[0027] SEQ ID NO. 2: the sequence shown is the nucleotide sequence of RPA primer CMN-RPA-R2.

[0028] SEQ ID NO. 3: the sequence shown is the nucleotide sequence of sgRNA target CMN-sgRNA-1.

[0029] SEQ ID NO. 4: the sequence shown is the nucleotide sequence of sgRNA target CMN-sgRNA-2.

[0030] SEQ ID NO. 5: the sequence shown is the nucleotide sequence of sgRNA target CMN-sgRNA-3.

[0031] SEQ ID NO.6: The sequence shown is the nucleotide sequence of the PCR primer CMN-PCR-F.

[0032] SEQ ID NO.7: The sequence shown is the nucleotide sequence of the PCR primer CMN-PCR-R.

[0033] SEQ ID NO.8: The sequence shown is the nucleotide sequence of the RPA primer CMN-RPA-F2.

[0034] SEQ ID NO.9: The sequence shown is the nucleotide sequence of RPA primer CMN-RPA-F3.

[0035] SEQ ID NO.10: The sequence shown is the nucleotide sequence of the RPA primer CMN-RPA-R1.

[0036] SEQ ID NO.11: The sequence shown is the nucleotide sequence of the RPA primer CMN-RPA-R3. Example

[0037] 1. Experimental Materials 1.1 Instruments and Equipment SynSor Portable Multi-Target Rapid Detection Device, XS-D-001 [Xunshi Biosciences]; QPCR Instrument - SLAN-96P [Shanghai Hongshi Medical Technology Co., Ltd.]; PCR Instrument (TC-S / 96 / G / H(b)BA) [Hangzhou Borui Technology Co., Ltd.]; Qubit 3.0 Fluorometer [Thermo Fisher Scientific]; Nanodrop 2000 Micro-volume Spectrophotometer [Thermo Fisher Scientific]; Metal Bath (DH100-2) [Hangzhou Ruicheng Instrument Co., Ltd.]

[0038] 1.2 Reagents SynSor AaCas 12b (C2c1) (XS-R-002) [Synsorbio]; SynSor DNA / RNA Isothermal Rapid Amplification Reagent (XS-R-101) [Synsorbio]; SynSor CRISPR ssDNA Reporter (12b-FAM) (XS-R-201) [Synsorbio]; SynSor sgRNA (XS-R-301) [Synsorbio]; Synsor CRISPR Single Target Detection Test Strip (FAM-Biotin) (XS-R-102) [Synsorbio]; Gold MIX (Green) (TSE101) [Beijing Qikeli Biotechnology Co., Ltd.]; The water used is UltraPure™ Distilled water, Dnase, Rnase, Free (10977-015) [Engelbrecht (Shanghai) Trading Co., Ltd.].

[0039] 2、Experimental method 2.1, sgRNA design and verification 2.1.1, sgRNA design According to the position of Clavibacter michiganensis subsp. Nebraskensis in Genbank index number CP132105.1 (2770567-2770788), the sgRNA of Clavibacter michiganensis subsp. Nebraskensis was designed by referring to GB / T 36840-2018 "Method for quarantine identification of Clavibacter michiganensis subsp. Nebraskensis", and the specific sequence information of the three sgRNAs designed is shown in Table 1.

[0040] Table 1 Information of sgRNA used

[0041] 2.1.2, sgRNA performance verification 1) PCR primer design According to the position of sgRNA design, the region within 100bp of sgRNA upstream and downstream was selected to design PCR primer, and the specific sequence information of the primer is shown in Table 2.

[0042] Table 2 Information of PCR specific primers used

[0043] 2) Template amplification verification According to the following system configuration solution, PCR amplification was carried out.

[0044] PCR amplification system: 1.1x gold MIX (Green) 42 μl; 10 μM upstream and downstream primers 2 μl each, template 2 μl, water 2 μl.

[0045] PCR reaction program: 98℃ pre-denaturation 2min; 98℃ denaturation 10s, 50℃ annealing 15s, 72℃ extension 10s, 30 cycles; final 72℃ terminal extension 5min; 10℃ preservation Qubit double-stranded DNA nucleic acid fluorescent dye concentration detection was performed on the amplification products of the target to be tested and the blank control. The concentration of the target to be tested should be higher than 2 ng / μl, and there should be a clear distinction from the concentration of the blank control.

[0046] 3) sgRNA verification The CRISPR system was prepared according to Table 3.

[0047] Table 3 CRISPR system preparation

[0048] 1 μl of about 10~100 ng of PCR amplification product was mixed with the CRISPR system in Table 3, and the FAM fluorescence signal was collected every minute in a qPCR instrument set at 43℃ for 15 min. According to the curve change of fluorescence signal, the performance or specificity of sgRNA was preliminarily verified. The endpoint of the fluorescence value curve of the target amplification product and the negative control should be significantly different.

[0049] The calculation formula of fluorescence growth rate is as follows: .

[0050] Where Fluorescence Slop represents the fluorescence growth rate, Rn represents the fluorescence signal at the nth minute, and R1 represents the fluorescence signal at the first minute of the negative control.

[0051] 2.2, RPA primer design The principle of RPA primer design is: within the detection target range, select a 30-35 base length fragment as the RPA primer candidate; to ensure stability and specificity, the GC content of the RPA primer should be between 40%-60%, and the RPA amplification fragment is usually 100-200 bp. Based on this primer design principle, 3 upstream and downstream primers were designed for RPA primer verification, and NCBI primer-BLAST was used for amplification coverage and specificity verification.

[0052] Based on the above principles, RPA specific primers for CMN were designed, as shown in Table 4: Table 4 RPA primer table

[0053] 2.3, Cube system detection system verification 2.3.1, Cube platform and chip technology principle SynsorCube-4X (hereinafter referred to as Cube) platform is a detection platform developed by Beijing Xunsi Technology Co., Ltd. which can simultaneously detect up to 4 samples of 4 targets. Based on RPA amplification, CRISPR detection, centrifugal microfluidic fully enclosed chip platform and intelligent photographing fluorescence detection system technology (, and combined with reagent dry powder storage process, it tries to realize a fully enclosed integrated nucleic acid rapid detection system, which can realize the rapid identification of target, and completely realize the detection mode of "sample in, result out". Figure 1

[0054] SynsorCube-4X microfluidic chip (hereinafter referred to as Cube chip) is designed and customized, which physically distinguishes sample loading channel, RPA amplification cavity and CRISPR detection cavity (Figure 2), avoiding cross interference between the three, so that the performance of each step can be fully played out, greatly improving the accuracy and sensitivity of the detection system for nucleic acid target identification; In order to further reduce the complexity of the detection system, solve the problem of differential centrifugation platform commonly used in the market, the flow channel siphon valve design is introduced in the Cube chip, which can realize the precise transfer of liquid under different centrifugation processes (, under the condition of constant speed of centrifugal module), further reducing the complexity and design cost of the whole detection system; As an important part of the detection system, the supporting equipment is an intelligent photographing fluorescence detection system that can perform multi-step centrifugation, incubation reaction, fluorescence signal collection, analysis and output, realizing the accurate reception and interpretation of the final detection fluorescence signal. Figure 3

[0055] 2.3.2, Cube platform reaction system configuration 1) Cube platform CRISPR system configuration and preparation process Due to the particularity of Cube platform, in-situ verification of CRISPR system is needed before verification. The CRISPR in-situ system is shown in Table 5.

[0056] Table 5 Cube process CRISPR detection system preparation

[0057] After the above CRISPR system preparation is completed, 10 μL of the system is added to reaction chamber 2, and the chip is placed in a 50℃ oven, and the system is dried for 1.5 h; After drying is completed, the chip is taken out, and after being covered with a film, it is placed in a sealed aluminum foil bag and stored in a low humidity environment.

[0058] ​​2) Cube platform CRISPR system verification stage RPA system configuration This part of the test first carries out the preparation of the target RPA amplification system. The final concentration of each component in the RPA system in this process is the same as that in section 3.2. The addition amount of RPA freeze-dried ball in the system is 1. The addition amount of each component in the RPA system in this process is shown in Table 6.

[0059] Table 6 Cube platform RPA system preparation

[0060] After mixing all the reaction systems in RPA, transfer all the systems to the Cube chip sample hole, then tightly paste the chip sealing film, and place the Cube chip in the Cube instrument. Start the instrument on the mobile phone for detection. After the reaction process is completed, the Cube chip detection results can be viewed on the mobile phone, and the detection results can be read.

[0061] 3) Cube platform whole process system configuration and preparation process The CRISPR reaction system configuration and preparation process in the whole process chip are shown in 1) of 2.3.2.

[0062] The addition amount of each component in the RPA system in this process is shown in Table 7. The components of the sample liquid in the Cube platform whole process are shown in Table 8.

[0063] Table 7 Cube platform whole process RPA primer system

[0064] During the preparation of the whole process chip, the CRISPR system in Table 5 and the RPA primer system in Table 7 are placed in reaction chamber 2 and reaction chamber 1 respectively, and the chip is placed in a 50°C oven. The system is dried for 1.5 h; after drying is completed, the chip is taken out, covered with a film, and placed in a sealed aluminum foil bag in a low humidity environment.

[0065] Table 8 Cube platform whole process sample liquid

[0066] After mixing all the sample liquid systems, transfer them to the Cube chip sample hole, then tightly paste the chip sealing film, and place the Cube chip in the Cube instrument. Start the instrument on the mobile phone for detection. After the reaction process is completed, the Cube chip detection results can be viewed on the mobile phone, and the detection results can be read.

[0067] 2.3.3, Chip detection process Use the prepared nucleic acid sample to verify this part.

[0068] The chip testing process is shown in Table 9, with a total testing time of approximately 28 minutes.

[0069] Table 9 POCT Instrument Experimental Procedure

[0070] 3. Experimental Results 3.1 sgRNA screening results The fluorescence intensity of the CRISPR reaction was measured for each sgRNA and its corresponding template amplification product. The fluorescence curves and fluorescence growth rate trends are shown in [the table below]. Figure 4 The fluorescence growth rate results are shown in Table 10. The analysis results show that the CMN target CMN-sgRNA-1 (SEQ ID NO.3) has good performance and can be used for subsequent RPA primer screening.

[0071] Table 10 Summary of Fluorescence Signal Changes in PCR Products

[0072] 3.2 RPA primer screening results Different combinations of RPA primers were screened by diluting plasmid samples to 1E4 copies / T and 1E3 copies / T, and a two-step RPA-CRISPR reaction was performed. The fluorescence curves and fluorescence growth rate trends of different primer sets in this procedure are shown in [the table below]. Figure 5 The effective amplification rates of each primer at each dilution gradient are shown in Table 11.

[0073] Table 11 Summary of Fluorescence Signal Changes in RPA Products

[0074]

[0075] CMN target primer validation results showed that CMN-RPA-F1 / R2 (SEQ ID NO.1 / SEQ ID NO.2) exhibited strong fluorescence signals under both high concentration (1E4 copies / T) and low concentration (1E3 copies / T) conditions. Figure 5 Therefore, CMN-RPA-F1 / R2 (SEQ ID NO.1 / SEQ ID NO.2) was selected for subsequent performance verification experiments.

[0076] 3.3 Construction and Validation of the Testing System 3.3.1 Validation of the sensitivity of the detection system Gradient dilution is performed using the plasmid template, and the gradient settings include 1E4 copies / T, 1E3 copies / T, 1E2 copies / T, 1E1 copies / T, and 1E0 copies / T. The target sensitivity verification is performed using the primers and sgRNA combination determined in 3.2, and the same CRISPR system. Each gradient is verified twice.

[0077] The amplification curve and fluorescence growth rate of CMN sensitivity verification Figure 6 ) 1E2 copies / T can be detected.

[0078] 3.3.2, detection system sensitivity confirmation After completing the sensitivity verification, 10 experimental repetitions are performed near the target detection limit to confirm the target sensitivity. According to the sensitivity of each target point verified in the early stage, 5E2 and 1E2 copies / T of CMN target are used. The amplification curve and fluorescence signal growth rate of CMN detection limit verification are shown in Figure 7 .

[0079] The sensitivity verification and sensitivity confirmation results of the RPA-CRISPR detection system of CMN show that CMN exhibits good stability in the detection limit and 10 repetitions near the detection limit. Therefore, the detection limit of CMN is 1E2 copies / T, and the detection sensitivity is better than that of the current common method.

[0080] 3.3.3, single-target chip CRISPR reagent in-situ drying feasibility verification The plasmid samples synthesized in the early stage are diluted, and the loading concentration is 1E4 copies / T. The chip preparation process in this process is 1) in 2.3.2, the loading system is 2) in 2.3.2, and the verification results of CMN are as follows Figure 8 The test results of this round show that CMN can produce strong fluorescence signals after CRISPR in-situ, indicating that the CRISPR in-situ performance of CMN meets the subsequent test requirements.

[0081] 3.3.4, single-target chip RPA and CRISPR double-system in-situ drying feasibility verification Based on the verification of CRISPR detection single-system in-situ in the early stage, RPA and CRISPR double-system in-situ drying tests are further carried out. This part uses nucleic acid samples diluted to 1E4 copies / T, and the chip test double-system drying process is 3) in 2.2.3, the CRISPR and RPA reagent in-situ system and the cube chip loading liquid system are shown in Table 5, Table 6 and Table 8. The test results of this round are shown in Figure 9The test results of this round show that the primer combination developed in the early stage has good detection results in the RPA and CRISPR double system in situ dried test. The CMN double system in situ can realize positive detection, which shows that the subsequent multi-target cube chip verification can be carried out.

[0082] To sum up, the present application develops an isothermal amplification detection method based on RPA and CRISPR for CMN disease targets in corn, which breaks away from the dependence on complex instrument equipment platform of traditional molecular biology methods, greatly simplifies the detection platform; through system establishment and verification, the overall detection time of the target is shortened to within half an hour, and there is still room for further reduction in the future; the present application also verifies the sensitivity, specificity and POCT platform of the target detection technology, and the results show that the detection method developed by the present application has good sensitivity and specificity, and the actual sample verification results are consistent with the theoretical results. The detailed detection limit and verification data of the target are as follows: The CMN target sgRNA is determined as CMN-sgRNA-1, which is matched with CMN-RPA-F1 / R2 for detection effect, and the minimum detection limit is 1E2 copies / T. The RPA primer set of the in-tube system of this target can be used for Cube chip test, and the sensitivity under the Cube chip detection process can reach 1E2 copies / T.

Claims

1. A reagent combination for detecting Clavibacter michiganensis subsp. nebraskense, characterized in that, Including specific RPA primer pairs and sgRNA; The RPA primer pair consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; the sgRNA is capable of targeting a specific sequence in the product amplified by the RPA primer pair, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. A kit for detecting maize wilt pathogen, characterized in that, Include: (1) The reagent combination according to claim 1; (2) Cas12b protein; (3) ssDNA reporter molecule, wherein one end of the reporter molecule is labeled with a fluorescent reporter group and the other end is labeled with a quencher group.

3. A method for detecting maize wilt pathogens (not for diagnostic purposes), characterized in that, Includes the following steps: S1. Using the nucleic acid of the sample to be tested as a template, perform RPA reaction using specific primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; S2, CRISPR detection reaction: The amplification product is mixed with the sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule and incubated at a constant temperature. S3. Detect the fluorescence signal and determine whether the sample to be tested contains maize wilt pathogen based on the fluorescence signal.

4. The detection method according to claim 3, characterized in that, The RPA reaction was carried out at 43°C for 15 minutes; the isothermal incubation was carried out at 43°C for 10 minutes.

5. The detection method according to claim 3, characterized in that, The RPA reaction, CRISPR detection reaction, and fluorescence signal detection are all automatically completed on a centrifugal microfluidic chip; the centrifugal microfluidic chip includes an RPA reaction chamber and a CRISPR detection chamber pre-positioned with the RPA primer pair and sgRNA from the reagent combination of claim 1, which are mutually isolated.

Images