System, kit and method for detecting fusarium moniliforme of fusarium moniliforme

Abstract

The invention provides a system, a kit and a method for detecting fusarium moniliforme of fusarium moniliforme of rice fusarium moniliforme. The system can accurately, quickly and efficiently detect weak positive and suspected positive samples with extremely low content of fusarium moniliforme of fusarium moniliforme of rice fusarium moniliforme, and has relatively high sensitivity and specificity, and the sensitivity can reach 1 copy / mu L and is far beyond the lower detection limit in the prior art. Therefore, the method can detect the fusarium moniliforme in early rice seeds, so that the rice bakanae disease is prevented.

Description

Technical Field

[0001] The invention belongs to the field of pathogen detection, and in particular relates to a system, a kit and a method for detecting rice seedling pathogen Fusarium fujikura. Background Art

[0002] Bakanae disease of rice, also known as leggy disease, is a worldwide disease. Since it was first reported in Japan in 1828, it has occurred in almost all rice-growing areas in the world. It is widely distributed in rice-growing areas in China, causing different degrees of damage, generally reducing production by 10%-20%, and up to 50% in severe cases. The main symptoms of bakanae disease of rice are: leggy growth, slender light green leaves, dwarfing, and seedling death in the seedling stage; root and stem base rot in the adult stage, and in severe cases, the whole plant dies or grows leggy, and grows upside down roots; after rice filling, the grains of seriously infected plants are infected with bacteria (pink or white mold layer). The pathogenic bacteria of rice bakanae disease are mainly Gibberella fujikuroi species complex, including Fusarium fujikuroi, F. verticillioides and F. proliferatum, with Fusarium fujikuroi being the main pathogen. Bakanae disease is a seed-borne disease, mainly transmitted through seeds, and secondly through soil, diseased plants, etc. At present, the prevention and control of bakanae disease is mainly achieved through various means such as chemical treatment and biological control. In addition to the above prevention and control measures, it is also necessary to carefully select and breed seeds that do not carry bakanae bacteria. Therefore, selecting disease-free seeds through early detection is a simple, easy, fast and effective preventive measure.

[0003] At present, the detection methods of rice seedling blight pathogen have been developed, such as morphological identification, mating type detection, fungal toxin detection, vegetative affinity classification, random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis, etc. However, most of these methods are time-consuming and labor-intensive, and even cannot accurately identify the pathogen.

[0004] At present, fluorescent quantitative PCR and ordinary PCR are relatively effective methods for detecting the rice seedling pathogen Fusarium fujikura. However, for samples with extremely low nucleic acid content, that is, Ct values ​​of 30-40, it is impossible to determine the positive and negative nature of the samples 100%. For example, Chinese patent CN 110117592A discloses a method for rapid detection of rice seedling pathogen Fusarium fujikura based on RPA, but its sensitivity concentration for detecting Fusarium fujikura hyphae is 0.8619ng / μL. Chinese patent CN 107022624A discloses a method for detecting rice seedling pathogen Fusarium fujikura using a loop-mediated isothermal amplification method, and the minimum detection concentration is 0.01ng / μL. However, early detection cannot be achieved for rice seeds with very small amounts of Fusarium fujikura, the rice seedling pathogen. Summary of the invention

[0005] In order to solve the problem of high detection limit of rice seedling disease Fusarium fujikura in the prior art, the present invention provides a system, a kit and a method for detecting rice seedling disease Fusarium fujikura, which can realize the detection of rice seedling disease Fusarium fujikura at an early stage, thereby preventing rice seedling disease.

[0006] The technical solution adopted by the present invention is: a system for detecting rice seedling pathogen Fusarium fujikura, the system comprising an RPA amplification system and a CRISPR / Cas12a detection system; the RPA amplification system comprises an RPA primer set for Fusarium fujikura; the CRISPR / Cas12a detection system comprises a Cas12a protein or a gene encoding it, a crRNA for Fusarium fujikura, and an ssDNA reporter system; the RPA primer set comprises sequences SEQ ID NO.1 and SEQ ID NO.2; the sequence of the crRNA is selected from any one of SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.9; the ssDNA reporter system comprises ssDNA, and one end of the ssDNA is labeled with a fluorescent reporter molecule or a CRISPR lateral flow test strip reporter molecule.

[0007] Table 1. RPA primers, crRNA and ssDNA reporter systems

[0008]

[0009] Preferably, one end of the ssDNA is labeled with a fluorescent reporter molecule, and the other end is labeled with a fluorescent quencher group.

[0010] Preferably, the fluorescent reporter molecule is selected from FAM, TET, JOE, VIC, HEX, ROX, TAMRA, Cy3, Cy3.5, Cy5, Cy5.5, OregonGreenTM, CALRedTM, Red640, Texas Red, Cyan500, Any one of Red610, biotin-binding material, Alexa 647, Alexa 555, 5-(2-aminoethyl)amino-1-naphthalenesulfonic acid, tetramethylrhodamine, tetramethylrhodamine isocyanate, fluorescein isocyanate, and x-rhodamine.

[0011] Preferably, the fluorescence quenching group is selected from any one of DDQ-I, DDQ-II, Dabcyl, Eclipse, Iowa BlackFQ, IowaBlack RQ, BHQ1, BHQ2, BHQ3, QSY7, QSY9, and QSY21.

[0012] Preferably, the ssDNA reporter system sequence is selected from any one of SEQ ID NO.10 or SEQ ID NO.11, more preferably SEQ ID NO.10.

[0013] Preferably, the sequence of the crRNA is SEQ ID NO.3.

[0014] Preferably, the system includes an RPA amplification system and a CRISPR / Cas12a detection system; the RPA amplification system includes an RPA primer set for Fujikura Fusarium with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; the CRISPR / Cas12a detection system includes a Cas12a protein or its encoding gene, a crRNA for Fujikura Fusarium with a sequence as shown in SEQ ID NO.3, and an ssDNA reporter system with a sequence as shown in SEQ ID NO.10. In a specific embodiment, the preferred system can perform efficient fluorescence detection on weakly positive and suspected positive samples with extremely low content of Fujikura Fusarium of rice seedling pathogen, and the sensitivity can reach 1copy / μL (i.e., 3.3×10 -7 ng / μL).

[0015] Preferably, the system includes an RPA amplification system and a CRISPR / Cas12a detection system; the RPA amplification system includes an RPA primer set for Fusarium fujikura, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2; the CRISPR / Cas12a detection system includes a Cas12a protein or its encoding gene, a crRNA for Fusarium fujikura, the sequence of which is shown in SEQ ID NO.3, and an ssDNA reporter system, the sequence of which is shown in SEQ ID NO.11. In a specific embodiment, the preferred system can perform efficient CRISPR lateral flow detection on weakly positive and suspected positive samples with extremely low content of Fusarium fujikura of rice seedling pathogen, and the sensitivity can reach 100copy / μL (i.e., 3.3×10 -4 ng / μL).

[0016] The present invention also provides an application of the system, which is any one of the following (1) to (4):

[0017] (1) Identifying or assisting in the identification of whether the pathogen to be tested is Fusarium fusokura;

[0018] (2) preparing a kit for identifying or assisting in identifying whether the pathogen to be tested is Fusarium fujikura;

[0019] (3) detecting whether the biological sample to be tested contains Fusarium fujikura;

[0020] (4) Prepare a kit for detecting whether a biological sample to be tested contains Fusarium fujikura.

[0021] The invention also provides a kit for detecting rice bakanae pathogen Fusarium fujikura, comprising the detection system.

[0022] Preferably, the kit further comprises at least one of a DNA extraction reagent, an RPA buffer, magnesium acetate, an RNase inhibitor, a CRISPR / Cas12a buffer, and a CRISPR lateral flow test strip.

[0023] The present invention also provides an application of the kit, which is any one of the following (1) to (2):

[0024] (1) Identifying or assisting in the identification of whether the pathogen to be tested is Fusarium fusokura;

[0025] (2) Detecting whether the biological sample to be tested contains Fusarium fujikura.

[0026] The present invention also provides a method for identifying or assisting in identifying whether the pathogen to be detected is Fusarium fujikura by using the system or the kit, comprising the following steps:

[0027] (1) Extracting DNA of the pathogen to be tested;

[0028] (2) performing RPA amplification on the DNA extracted in step (1) using the RPA amplification system to obtain an RPA amplification product;

[0029] (3) using the CRISPR / Cas12a detection system to perform a trans-cleavage reaction on the RPA amplification product obtained in step (2) to obtain a reaction product;

[0030] (4) Performing corresponding fluorescence detection or CRISPR lateral chromatography detection on the reaction products according to the ssDNA reporter system.

[0031] The present invention also provides a method for detecting whether a biological sample to be tested contains Fusarium fujikura by using the system or the kit, comprising the following steps:

[0032] (1) Extracting DNA from the biological sample to be tested;

[0033] (2) performing RPA amplification on the DNA extracted in step (1) using the RPA amplification system to obtain an RPA amplification product;

[0034] (3) using the CRISPR / Cas12a detection system to perform a trans-cleavage reaction on the RPA amplification product obtained in step (2) to obtain a reaction product;

[0035] (4) Performing corresponding fluorescence detection or CRISPR lateral chromatography detection on the reaction products according to the ssDNA reporter system.

[0036] Preferably, a 50 μL RPA amplification reaction system includes: 29.5 Primer free rehydration buffer, 2.410 μM RPA-F, 2.410 μM RPA-R, 1 uL DNA sample to be tested, 12.2 μL H2O, and 2.5 μL 280 mM magnesium acetate. The components other than 280 mM MgOAc are mixed to obtain a mixed solution, the volume of which is 47.5 μL, and the mixed solution is transferred to a TwistAmpTM Basic reaction tube containing lyophilized enzyme powder, and the lyophilized enzyme powder is blown with a pipette until it is completely dissolved, and then 2.5 μL 280 mM MgOAc is added and mixed, and the TwistAmpTM Basic reaction tube is placed in a 39°C constant temperature incubator for 20 minutes to complete RPA amplification and obtain RPA amplification products.

[0037] Preferably, 20 μL of the trans-cleavage reaction system includes: 1 μL 1 μM LbCas12a, 1 μL 5 μM crRNA, 1 μL RPA amplification product, 0.5 μL 10 μM ssDNA reporter system, and 2 μL 10×TOLO Buffer 3. The components were mixed and incubated in a real-time PCR instrument (QuantStudio 6Flex) for 60 minutes, and the fluorescence signal was measured every 30 seconds.

[0038] Preferably, when using an ssDNA reporter system labeled with a CRISPR lateral flow assay test strip reporter molecule, the reaction product is subjected to CRISPR lateral flow assay using the CRISPR lateral flow assay test strip; if the CRISPR lateral flow assay test strip test result is positive, the pathogen to be tested is Fusarium fujikura, or the biological sample to be tested contains Fusarium fujikura.

[0039] Preferably, when using an ssDNA reporter system labeled with a fluorescent reporter molecule, the reaction product is fluorescently detected using a fluorescence detection device; if the fluorescence detection device detects a rising fluorescence signal, or a color change reaction is observed under a fluorescent lamp, the pathogen to be tested is Fusarium fujikura, or the biological sample to be tested contains Fusarium fujikura.

[0040] Beneficial effects of the present invention:

[0041] The embodiment of the present invention provides a system, a kit and a method for detecting the rice seedling pathogen Fujikura Fusarium, which can accurately and quickly detect the rice seedling pathogen Fujikura Fusarium, and has high sensitivity and specificity. First, the sequence of the RPA primer set used in the embodiment of the present invention is relatively short. Generally, the lengths of the first upstream primer and the first downstream primer are about 35 bases, respectively. On the basis of ensuring the amplification effect, the sequence lengths of the first upstream primer RPA-F and the first downstream primer RPA-R provided in the embodiment of the present invention are controlled at 32 bases, thereby reducing the synthesis cost of the first upstream primer and the first downstream primer. Secondly, the 7 different crRNA sequences designed by the present invention also have huge differences in the effect of the trans-splicing reaction. Only crRNA1, crRNA2, and crRNA7 can have a better effect in the trans-splicing reaction, produce a relatively excellent fluorescence signal, and react under the action of crRNA1 for 5 to 10 minutes to reach a higher fluorescence value, when the reaction is 40 minutes, the highest fluorescence value can be reached, indicating that the quality of crRNA1 is high. In a preferred embodiment provided by the present invention, the RPA primer set shown in SEQ ID NO.1 and SEQ ID NO.2 and the crRNA1 shown in SEQ ID NO.3 can be used to efficiently detect weak positive and suspected positive samples with extremely low content of rice seedling pathogen Fusarium fusariosporium, and the sensitivity can reach 1 copy / μL (i.e., 3.3×10 -7 ng / μL), 1000 copies / μL (i.e. 3.3×10 -4 ng / μL) far exceeds the detection limit in the prior art, producing unexpected effects, and can also increase the fluorescence value by increasing the reaction time, thereby improving the accuracy of the detection results, which enables the method to detect the rice seedling blight pathogen Fujikura Fusarium in early rice seeds, thereby preventing rice seedling blight. In summary, obtaining the crRNA with good specificity, sensitivity, and compatibility shown in SEQ ID NO.1 of the present application and combining it with the RPA primers shown in SEQ ID NO.1 to 2 with good amplification efficiency, and then obtaining the detection system with good sensitivity and specificity for which the present invention is requested to protect is not only obtainable through logical analysis, reasoning, or limited experiments based on the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings are used to provide a further understanding of the technical solution of the present invention and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present invention and do not constitute a limitation on the technical solution of the present invention.

[0043] Figure 1This is a fluorescence value graph of the 7 crRNAs provided in Example 1 of the present invention for detecting the rice seedling pathogen Fusarium fujikura for 0-60 minutes.

[0044] Figure 2 This is the agarose gel electrophoresis diagram provided in Example 2 of the present invention.

[0045] Figure 3 This is a specific diagram of CRISPR / Cas12a provided in Example 2 of the present invention for detecting the rice seedling pathogen Fusarium fujikura.

[0046] Figure 4 This is a fluorescence detection concentration diagram of the CRISPR / Cas12a system provided in Example 3 of the present invention.

[0047] Figure 5 This is a graph showing the concentration detection of Fusarium fujikura provided by Example 3 of the present invention.

[0048] Figure 6 This is a CRISPR lateral chromatography detection concentration diagram of the CRISPR / Cas12a system provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0049] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0050] Example 1: System and method for detecting rice bakanae pathogen Fusarium fujikura

[0051] This embodiment provides a system for detecting the rice seedling pathogen Fusarium fusokura, including: an RPA amplification system and a CRISPR / Cas12a detection system; the RPA amplification system includes an RPA primer set for Fusarium fusokura having a sequence as shown in SEQ ID NO.1 and SEQ ID NO.2; the CRISPR / Cas12a detection system includes Cas12a protein (purchased from Nanjing Youqing Biotechnology Co., Ltd.), crRNA1 to 7 for Fusarium fusokura having a sequence as shown in SEQ ID NO.3 to 9, respectively (all synthesized by Sangon Biotech (Shanghai) Co., Ltd.), and a ssDNA reporter system having a sequence as shown in SEQ ID NO.10.

[0052] 1. Extraction of DNA from Fusarium fujikuroi: Take a 200 μL centrifuge tube, add 8 μL ddH2O (double distilled water) and 1 μL Mg-free 2+ 10×PCR Buffer was added, and Fusarium fujikura, a pathogen of rice seedlings, was picked up and put into the centrifuge tube. The centrifuge tube was frozen in liquid nitrogen for 1 min, and then placed at 85°C for 2 min. 1 μL of 10 mg / mL proteinase K was added to the centrifuge tube, and the tube was gently mixed by pipetting and centrifuged. The centrifuge tube was placed at 56°C for 15 min and 95°C for 10 min, and the supernatant was aspirated to obtain DNA of Fusarium fujikura, and the supernatant was used as a template for amplifying DNA.

[0053] 2. RPA amplification reaction: The RPA amplification system is shown in Table 2, wherein the sample to be tested is the rice seedling pathogen Fusarium fujikura DNA obtained in step 1.

[0054] Table 2. RPA amplification system

[0055]

[0056] The components except 280 mM MgOAc were mixed to obtain a mixed solution with a volume of 47.5 μL. The mixed solution was transferred to a TwistAmpTM Basic reaction tube containing lyophilized enzyme powder, and pipetted with a pipette until the lyophilized enzyme powder was completely dissolved. Then, 2.5 μL 280 mM MgOAc was added and mixed. The TwistAmpTM Basic reaction tube was placed in a 39°C constant temperature incubator and cultured for 20 min to complete RPA amplification and obtain RPA amplification products.

[0057] 3. Trans-cleavage reaction and fluorescence detection: Mix the components of the CRISPR / Cas12a detection system according to Table 3, incubate in a real-time PCR instrument (QuantStudio 6Flex) for 60 minutes, and measure the fluorescence signal every 30 seconds. The measurement results are as follows Figure 1 shown.

[0058] Table 3 CRISPR / Cas12a detection system

[0059]

[0060] Depend on Figure 1 It can be seen that in this embodiment, the fluorescence values ​​under the action of crRNA1, 2, and 7 are relatively high, and crRNA1 can reach a relatively high fluorescence value after reacting for 5-10 minutes, and the highest fluorescence value can be reached after reacting for 40 minutes, indicating that the quality of crRNA1 is relatively high.

[0061] Example 2: Detection of the specificity of the rice bakanae pathogen Fusarium fujikura system

[0062] The detection system and detection method provided in Example 1 were used to detect ddH2O, three rice seedling pathogens Fusarium fujikuroi, three rice seedling pathogens F. verticillioides, and two rice seedling pathogens F. proliferatum. In this example, the negative control was ddH2O. The DNA of the rice seedling pathogens Fusarium fujikuroi, F. verticillioides, and F. proliferatum were extracted using the rice seedling pathogen Fusarium fujikuroi DNA extraction method in Example 1. At the same time, the RPA amplification products corresponding to each sample were detected by 1% agarose gel electrophoresis, and the detection results are shown in the figure. Figure 2 The measurement results of fluorescence detection under trans-cleavage reaction are shown in Figure 3 shown.

[0063] Depend on Figure 3 It can be seen that the detection system provided in Example 1 of the present invention has a high specificity for the rice seedling pathogen Fusarium fujikura.

[0064] Example 3: Sensitivity of the system for detecting the rice bakanae pathogen Fusarium fujikura

[0065] The DNA of the rice seedling pathogen Fusarium fusarifolia extracted in Example 1 was diluted to 10 10 -10 0 The concentration of copies / μL was then detected by the detection system and agarose gel electrophoresis provided in the embodiments of the present invention. The ssDNA reporter system (SEQ ID NO.10 or SEQ ID NO.11) labeled with a fluorescent reporter molecule or a CRISPR lateral flow detection test strip reporter molecule provided by the present invention was used to perform corresponding fluorescence detection or CRISPR lateral flow detection.

[0066] Depend on Figure 4 , Figure 6 It can be seen that the detection system provided by the embodiment of the present invention can detect at least 1 copy / μL ( Figure 4 , fluorescence detection), 10 3 copies / μL( Figure 6 , CRISPR lateral flow assay). The sensitivity of the CRISPR lateral flow assay is comparable to the detection limit of 10 3The concentration of copies / μL is consistent, but the reporting method is simpler and more intuitive, without the need for complex experimental operations. The detection system that reports by fluorescence has a sensitivity 1000 times higher than the fluorescence detection method, and far exceeds the detection limit of the existing technology, producing unexpected results. In addition, the fluorescence value can be increased by increasing the reaction time, thereby improving the accuracy of the detection results. This method can detect the rice seedling pathogen Fusarium fujikura in early rice seeds, thereby preventing rice seedling disease.

[0067] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A system for detecting rice seedling pathogen Fusarium fujikura, characterized in that: The system includes an RPA amplification system and a CRISPR / Cas12a detection system; The RPA amplification system includes an RPA primer set for Fusarium fujikura; The CRISPR / Cas12a detection system includes Cas12a protein or its encoding gene, crRNA for Fusarium fujikura, and ssDNA reporting system; The RPA primer set includes sequences SEQ ID NO.1 and SEQ ID NO.2; The sequence of the crRNA is selected from any one of SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.9; The ssDNA reporter system comprises ssDNA, one end of which is labeled with a fluorescent reporter molecule or a CRISPR lateral flow test strip reporter molecule.

2. The system according to claim 1, characterized in that One end of the ssDNA is labeled with a fluorescent reporter molecule, and the other end is labeled with a fluorescent quencher group; The fluorescent reporter molecule is selected from FAM, TET, JOE, VIC, HEX, ROX, TAMRA, Cy3, Cy3.5, Cy5, Cy5.5, OregonGreenTM, CALRedTM, Red640, Texas Red, Cyan500, Any one of Red610, biotin-binding material, Alexa 647, Alexa 555, 5-(2-aminoethyl)amino-1-naphthalenesulfonic acid, tetramethylrhodamine, tetramethylrhodamine isocyanate, fluorescein isocyanate, and χ-rhodamine; The fluorescence quenching group is selected from any one of DDQ-I, DDQ-II, Dabcyl, Eclipse, Iowa Black FQ, Iowa BlackRQ, BHQ1, BHQ2, BHQ3, QSY7, QSY9, and QSY21.

3. The system according to claim 1, characterized in that The ssDNA reporter system sequence is selected from any one of SEQ ID NO.10 or SEQ ID NO.

11.

4. The system according to claim 1, characterized in that The sequence of the crRNA is SEQ ID NO.3; The ssDNA reporter system sequence is SEQ ID NO.

10.

5. Use of the system according to any one of claims 1 to 4, characterized in that: Any one of the following (1) to (4): (1) Identifying or assisting in the identification of whether the pathogen to be tested is Fusarium fusokura; (2) preparing a kit for identifying or assisting in identifying whether the pathogen to be tested is Fusarium fujikura; (3) detecting whether the biological sample to be tested contains Fusarium fujikura; (4) Prepare a kit for detecting whether a biological sample to be tested contains Fusarium fujikura.

6. A kit for detecting the rice seedling pathogen Fusarium fujikura, characterized in that: The invention comprises the detection system described in any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that It also includes at least one of a DNA extraction reagent, an RPA buffer, magnesium acetate, an RNase inhibitor, a CRISPR / Cas12a buffer, and a CRISPR lateral flow test strip.

8. Use of the kit according to claim 6, characterized in that, Any of the following (1) to (2): (1) Identifying or assisting in the identification of whether the pathogen to be tested is Fusarium fusokura; (2) Detecting whether the biological sample to be tested contains Fusarium fujikura.

9. A method for identifying or assisting in identifying whether a pathogen to be detected is Fusarium fujikura using the system according to any one of claims 1 to 4 or the kit according to claim 6, comprising the following steps: (1) Extracting DNA of the pathogen to be tested; (2) performing RPA amplification on the DNA extracted in step (1) using the RPA amplification system to obtain an RPA amplification product; (3) using the CRISPR / Cas12a detection system to perform a trans-cleavage reaction on the RPA amplification product obtained in step (2) to obtain a reaction product; (4) Performing corresponding fluorescence detection or CRISPR lateral chromatography detection on the reaction products according to the ssDNA reporter system.

10. A method for detecting whether a biological sample to be tested contains Fusarium fujikura using the system according to any one of claims 1 to 4 or the kit according to claim 6, comprising the following steps: (1) Extracting DNA from the biological sample to be tested; (2) performing RPA amplification on the DNA extracted in step (1) using the RPA amplification system to obtain an RPA amplification product; (3) using the CRISPR / Cas12a detection system to perform a trans-cleavage reaction on the RPA amplification product obtained in step (2) to obtain a reaction product; (4) Performing corresponding fluorescence detection or CRISPR lateral chromatography detection on the reaction products according to the ssDNA reporter system.

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