Method for detecting phomopsis helianthi based on RPA / CRISPR-Cas12a

Abstract

The invention belongs to the field of agriculture and plant quarantine, and particularly relates to a method for detecting phomopsis helianthi based on RPA / CRISPR-Cas12a, which comprises the following steps: S1, designing a detection composition: designing a composition for detecting phomopsis helianthi, the composition comprising a specific primer RPA-F / R nucleic acid sequence, a specific crRNA nucleic acid sequence and probes FL-ssDNA and LF-ssDNA; s2, extracting DNA: extracting the genome DNA of the diseased tissue of the sunflower to be detected; s3, DNA isothermal amplification: taking the genome DNA obtained in the step S1 as a template, adding a specific primer RPA-F1 / R1 into the template, and carrying out RPA amplification to obtain an amplification product; s4, CRISPR / Cas12a reaction: the amplification product obtained in the S2 and the crRNA are respectively mixed with FL-ssDNA and LF-ssDNA probes for a cleavage reaction, and a reaction product FL and a reaction product LF are obtained; and S5, visual detection: respectively placing the reaction product FL and the reaction product LF in a fluorescent PCR instrument and on a Cas12 special detection test strip for detection. The method is based on an RPA / CRISPR-Cas12a technology, by designing a specific primer and a crRNA nucleic acid sequence, the detection duration of the phomopsis helianthi is greatly shortened, the detection sensitivity is improved, and the method has very high use value in detection scenes such as customs and the like.

Description

Technical Field

[0001] The present invention belongs to the fields of agriculture and plant quarantine, and particularly relates to a method for detecting Diaporthe helianthi using RPA / CRISPR-Cas12a. Background Art

[0002] Diaporthe helianthi Muntanola-Cvetkovic Mihaljcevic et Petrov is a highly destructive fungal pathogen on sunflowers and is listed in the list of quarantine pests for imported plants in China. This pathogen can infect all above-ground organs of sunflower plants, causing the stems to dry out and break easily, the flower disks to develop poorly, and unable to form mature seeds. In severe cases, it can cause the death of the entire plant, resulting in irreparable economic losses. Currently, this disease has not occurred in China. China imports a large amount of sunflowers from abroad every year, and the risk of this disease being introduced has increased significantly.

[0003] Currently, in the daily inspection at customs ports, the quarantine and identification of sunflower stem canker are carried out according to the national standard of the People's Republic of China, "Quarantine and Identification Method for Sunflower Stem Canker" (GB / T 31792-2015). The detection methods for disease identification include isolation and culture, conventional PCR, fluorescence PCR detection and other techniques, including sub-inspection, selection of diseased tissues for isolation and culture, morphological observation, nucleic acid extraction, amplification using corresponding primers, sequencing and other methods for detection. Among them, the traditional morphological identification method is very empirical and requires isolation and purification. Due to the slow growth of the pathogen and the often concomitant co-infection with other pathogens, it is difficult to isolate and purify the pathogen, and the identification takes a long time; the detection effect of conventional PCR for seed samples with a low pathogen-carrying rate is not ideal, and false negatives are prone to occur; real-time fluorescence quantitative PCR has the advantages of being not easily contaminated, simple to operate, and highly sensitive, but it requires special instruments and takes a long time. These commonly used methods must be operated by professionals in the laboratory, which cannot meet the high-efficiency supervision effectiveness and customs clearance efficiency of the customs, and cannot be used for on-site detection. There is an urgent need to develop a more suitable, low-cost, sensitive, specific and simple detection method for on-site and efficient customs clearance for Diaporthe helianthi. Summary of the Invention

[0004] Aiming at the problems of long time consumption and easy occurrence of false negatives in the current process of customs quarantine for imported sunflower stem canker, the present invention provides a method for detecting Diaporthe helianthi using RPA / CRISPR-Cas12a. This method is based on the RPA / CRISPR-Cas12a detection technology. By designing specific primers and crRNA, the detection accuracy of Diaporthe helianthi is greatly improved, and the detection time is reduced. The specific technical solutions are as follows:

[0005] First, the present invention provides a method for detecting *Diaporthe helianthi* based on RPA / CRISPR-Cas12a, comprising the following steps:

[0006] S1 Design of detection composition: Design a composition for detecting *Diaporthe helianthi*, which includes the specific primer RPA-F / R nucleic acid sequence, specific crRNA nucleic acid sequence, and probes FL-ssDNA and LF-ssDNA as described in claim 1.

[0007] S2 DNA extraction: Extract the genomic DNA of the diseased tissue of the sunflower to be detected.

[0008] S3 Isothermal DNA amplification: Using the genomic DNA obtained in step S1 as a template, add the specific primers RPA-F1 / R1 to perform RPA amplification to obtain an amplification product.

[0009] S4 CRISPR / Cas12a reaction: Mix and cut the amplification product obtained in S2 and crRNA with the probes FL-ssDNA and LF-ssDNA respectively to obtain the reaction product FL and the reaction product LF.

[0010] S5 Visual detection: Place the reaction product FL and the reaction product LF in a fluorescence PCR instrument and a Cas12 special detection test strip respectively for detection.

[0011] Furthermore, step S1 includes the following steps: Select a partial sequence of the translation elongation factor (EF-1α) gene of *Diaporthe helianthi* (Genbank: ON146064.1) as the object for primer design, design the specific primer RPA-F / R nucleic acid sequence that meets the RPA reaction conditions and is within the RPA primer amplification fragment, design the crRNA nucleic acid sequence, and design the probe sequences of FL-ssDNA and LF-ssDNA according to the Cas12a protease recognition site TTTN.

[0012] Furthermore, step S2 includes the following steps: Put the diseased tissue to be detected into ddH 2 O, mix well, incubate at 100 °C for 10 min, then use a DNA extraction kit to extract the DNA of the diseased tissue, and quantify the concentration of the extracted DNA using a spectrophotometer.

[0013] Furthermore, the reaction in step S3 includes the following system: 29.4 μL of reaction buffer A, 2 μL of 10 μmol / L upstream primer (RPA-F), 2 μL of 10 μmol / L downstream primer (RPA-R), 5 μL of DNA template, and finally add 2.5 μL of magnesium acetate solution, and the remaining is made up with DEPC-H 2 O.

[0014] Furthermore, the isothermal amplification reaction condition in step S3 is incubation at 38°C for 30 min.

[0015] Furthermore, the CRISPR / Cas12a reaction in step S4 includes the following system: DEPC-H 2 O 12.5 μL, 20 U / μL RNase inhibitor 1 μL, 10×Cas12a Buffer 2 μL, 10 μmol / L LbCas12a 1 μL, 10 μmol / L crRNA 1 μL, 10 μmol / L FL-ssDNA Reporter or LF-ssDNA Reporter 1 μL, RPA amplification product 2 μL.

[0016] Furthermore, step S5 includes the following steps:

[0017] S51 Fluorescence detection: Put the reaction product FL into a real-time fluorescence PCR instrument and react at 38°C for 30 min. After the reaction, irradiate the product under a blue light. If there is no luminescence in the product, the result is negative. If the product emits green fluorescence, the result is positive;

[0018] S52 Test strip detection: Incubate the reaction system in S4 at 38°C for 20 min to obtain the reaction product LF. Dilute the reaction product LF by 10 times and mix well. Immerse the absorbent pad end of the Cas12 special detection test strip into the diluted reaction product LF, and observe whether the quality control line and the detection line on the test strip appear; if only one quality control line appears, the reaction result is negative. If both the quality control line and the detection line appear or the detection line appears alone, the reaction result is positive.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The present invention establishes a method for detecting the pathogen of sunflower stem canker based on RPA / CRISPR-Cas12a. By using the self-designed specific primer sequence and the crRNA nucleic acid sequence with high cleavage efficiency, it can specifically detect the pathogen of sunflower stem canker, improve the sensitivity of detecting trace amounts of the pathogen of sunflower stem canker, and the detection sensitivity can reach 37 ag / μL in the fluorescence method. At the same time, the detection method of the present invention is efficient and rapid, and it only takes 80 min from sample preparation to obtaining the detection result, realizing the rapid and sensitive molecular detection of the pathogen of sunflower stem canker and meeting the current rapid customs clearance requirements at ports.

[0021] 2) The method established in the present invention is simpler in operation process and more intuitive in results compared with the commonly used PCR and real-time fluorescence quantitative PCR techniques in the laboratory. It only needs to observe the lateral flow test strip or the luminescence of the system under blue light irradiation to make a judgment. In this study, the RPA amplification and CRISPR-Cas12a cleavage systems are carried out in two steps. These two techniques have lower temperature requirements and can react rapidly at 37 - 42 °C with a large amplification amount. There is a risk of sample contamination during the actual operation when opening the lid to sample. Therefore, the integration of RPA amplification and CRISPR-Cas12a cleavage reactions can be used as the subsequent research direction to reduce the number of times of opening the lid during the reaction, reduce the possibility of sample contamination, and further improve the rapid diagnostic technique for Didymella exigua. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the experimental result diagram in Experimental Example 1 of a method for detecting Didymella exigua on sunflower stems based on RPA / CRISPR-Cas12a of the present invention;

[0023] Figure 2 It is the experimental result diagram in Experimental Example 2 of a method for detecting Didymella exigua on sunflower stems based on RPA / CRISPR-Cas12a of the present invention;

[0024] Figure 3 It is the experimental result diagram in Experimental Example 3 of a method for detecting Didymella exigua on sunflower stems based on RPA / CRISPR-Cas12a of the present invention;

[0025] Figure 4 It is the experimental result diagram in Experimental Example 4 of a method for detecting Didymella exigua on sunflower stems based on RPA / CRISPR-Cas12a of the present invention;

[0026] Figure 5 It is the process schematic diagram of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments.

[0028] Embodiment 1

[0029] As shown in the Figure 5 accompanying drawings, this embodiment provides a method for detecting Didymella exigua on sunflower stems based on RPA / CRISPR-Cas12a, including the following steps:

[0030] S1 Design of detection composition: Design a composition for detecting D. helianthi, which includes the specific primer RPA-F / R nucleic acid sequence, specific crRNA nucleic acid sequence, and probes FL-ssDNA and LF-ssDNA as described in claim 1;

[0031] Specifically, screen the published gene sequences of D. helianthi in the NCBI database (National Center for Biotechnology Information), select a partial sequence of the translation elongation factor (EF-1α) gene (Genbank: ON146064.1) as the object for primer design, and design specific primers RPA-F / R that meet the RPA reaction conditions. Within the RPA primer amplification fragment, design the crRNA nucleic acid sequence. The complete crRNA sequence consists of a stem-loop "5′-UAAUUUCUACUAAGUGUAGAU-3′" that ensures the cleavage function of Cas12a and a 23-base sequence after the TTTN base in the PAM region of the target DNA sequence. In the experiment, the RPA primers, crRNA, and single-stranded reporter molecules were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific primer sequences are shown in Table 1.

[0032] Table 1 Sequences of RPA primers, crRNA, and single-stranded reporter molecules

[0033]

[0034] S2 DNA extraction: Extract the genomic DNA of the diseased sunflower tissue to be detected;

[0035] Specifically, to meet the actual detection requirements, a rapid extraction method is used to prepare DNA. Put the sample with D. helianthi bacteria into 300 μL of ddH 2 O, mix well, incubate at 100 °C for 10 min, and then collect the supernatant as the crude extracted DNA for rapid detection. Use the Plant Genomic DNA Kit to extract the DNA from the supernatant. After extraction, quantify the concentration of the extracted DNA using a spectrophotometer. Store the extracted DNA at -20 °C for subsequent use.

[0036] S3 Isothermal amplification of DNA: Using the genomic DNA obtained in step S1 as a template, add specific primers RPA-F1 / R1 to it for RPA amplification to obtain an amplification product;

[0037] Specifically, for RPA amplification, referring to the instructions of the AmpFuture DNA isothermal rapid amplification kit, the reaction system is a total of 50 μL. Add 29.4 μL of reaction buffer A, 2 μL of 10 μmol / L upstream primer RPA-F, 2 μL of 10 μmol / L downstream primer RPA-R, 5 μL of DNA template to the reaction tube containing the lyophilized enzyme powder component. Finally, add 2.5 μL of magnesium acetate solution, and finally add 9.1 μL of ddH2O to make up to 50 μL. Mix the solution in the reaction tube thoroughly, and incubate the reaction tube in a 38°C water bath for 30 min to obtain the amplification product.

[0038] S4 CRISPR / Cas12a reaction: Mix the amplification product obtained in S2 and crRNA with FL-ssDNA and LF-ssDNA probes respectively for cleavage reaction to obtain reaction product FL and reaction product LF;

[0039] Specifically, the CRISPR / Cas12a reaction system library has the following components: DEPC-H 2 O 12.5 μL, 20 U / μL RNase inhibitor 1 μL, 10×Cas12a Buffer 2 μL, 10 μmol / L LbCas12a 1 μL, 10 μmol / L crRNA 1 μL, 10 μmol / L FL-ssDNA Reporter or LF-ssDNA Reporter 1 μL, RPA amplification product 2 μL. After all components are added, mix well and incubate at 38°C.

[0040] S5 Visual detection: Place reaction product FL and reaction product LF in a fluorescence PCR instrument and on a Cas12 special detection test strip respectively for detection.

[0041] In the present invention, visual detection includes fluorescence PCR detection and Cas12 special detection test strip detection. During fluorescence PCR detection, put reaction product FL into a real-time fluorescence PCR instrument and react at 38°C for 30 min. After the reaction is completed, place the product under a blue light. If the product does not emit light, the result is negative. If the product emits green fluorescence, the result is positive.

[0042] When using the Cas12 special detection test strip for detection, incubate the reaction system in S4 at 38°C for 20 min to obtain reaction product LF. Dilute reaction product LF 10 times and mix well. Immerse the absorbent pad end of the Cas12 special detection test strip into the diluted reaction product LF, and observe whether the quality control line and the detection line on the test strip appear; if only one quality control line appears, the reaction result is negative. If both the quality control line and the detection line appear or only the detection line appears alone, the reaction result is positive.

[0043] Experimental Example 1: Specificity Experiment of crRNA Nucleic Acid Sequence

[0044] Experimental method: A total of 3 crRNA nucleic acid sequences and a blank control group were designed. According to the detection method of Example 1, they were added to the RPA / CRISPR-Cas12a fluorescence method system respectively, and the fluorescence intensity was measured under a real-time fluorescence detector. The nucleic acid sequence of crRNA1 among the 3 crRNA nucleic acid sequences is:

[0045] 5′-UAAUUUCUACUAAGUGUAGAUAGUGCGGGUGCGGGUACGGGUGC-3′, SEQ ID NO: 4,

[0046] The nucleic acid sequence of crRNA3 is:

[0047] 5′-UAAUUUCUACUAAGUGUAGAUCACCCCUCCCUCUGGAUUUUCCA-3′, SEQ ID NO: 5,

[0048] crRNA2 is the crRNA in Example 1, and Control is the blank control.

[0049] The experimental results are as shown in the appendix Figure 1 It can be seen from the experimental results that when using the crRNA nucleic acid sequence in the present invention for fluorescence intensity detection, the detected fluorescence intensity is the strongest, reaching the highest value within 30 min, which is higher than that of crRNA1 and crRNA3, indicating that crRNA has a high cleavage efficiency in the system and is suitable for the reaction system of the present invention.

[0050] Experimental Example 2: Specificity Verification of RPA / CRISPR-Cas12a Detection System

[0051] Experimental method: According to the detection method of Example 1, using the primers and crRNA nucleic acid sequences in Example 1, the strains prepared in Table 2 below were detected by RPA / CRISPR-Cas12a, and the detection results are as shown in the appendix Figure 2 shown. In the appendix Figure 2 In Figure A is the specificity verification by fluorescence method; Figure B is the specificity verification by test strip method; in Figures A and B, Treatments 1-4 are the strains carrying sunflower stem canker, Treatments 5-11 are the control strains carrying the corresponding pathogens in Table 2, and Treatment 12 is the blank control.

[0052] Table 2 Information of Test Strains

[0053]

[0054] Analysis of experimental results: In the fluorescence method detection (appendix Figure 2-A), only 4 strains carrying the pathogen of sunflower stem canker showed green fluorescence visible to the naked eye under blue light irradiation, and no luminescence was observed in other tested strains. In the test strip method (attached Figure 2 -B), the test results of the 4 strains carrying the pathogen of sunflower stem canker were all positive, and the test results of other tested strains were negative. The test results were consistent with those of the fluorescence method, indicating that this system is specific for detecting the pathogen of sunflower stem canker.

[0055] Experimental Example 3: Detection experiment of simulated samples

[0056] Experimental method: According to the experimental method of Example 1, 0.01 g, 0.02 g, 0.04 g, 0.08 g, and 0.16 g of mycelia of the pathogen of sunflower stem canker were respectively put into 0.02 g of sterile sunflower sample powder to obtain crude extracted DNA, and the simulated sunflower seed-borne experiment was combined with the RPA-CRISPR / Cas12a detection system for the pathogen of sunflower stem canker to conduct the test. The test results are as attached Figure 3 shown. Among them, Figure A is the detection of simulated samples by the test strip method; Figure B is the detection of simulated samples by the fluorescence method; Figure C is the test result of real-time fluorescence quantitative PCR for simulated samples. The labeled amount of bacteria carried is shown on Figure A.

[0057] Experimental results: The detection results of the RPA / CRISPR-Cas12a fluorescence method and the test strip method (attached Figure 3 -A, B) show that the test results of the bacterium-carrying samples were all positive, and the test results of the sterile sunflower samples were negative; at the same time, they were consistent with the results of the real-time fluorescence quantitative detection method used in the quarantine and identification method for the pathogen of sunflower stem canker (GB / T 31792-2015) (attached Figure 3 -C), indicating that this system can accurately and rapidly detect the pathogen of sunflower stem canker.

[0058] Experimental Example 4: Sensitivity experiment

[0059] Experimental method: In the sensitivity determination experiment, plasmid standard products were used for determination. According to the instructions of the pGM-T product cloning kit, the RPA amplification product fragment was ligated into the pGM-T vector, and then the vector containing the target fragment was transformed into DH5α competent cells. After the transformation was completed, the reference plasmid was obtained and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification. After confirming that the plasmid fragment was consistent with the expected fragment, the plasmid was purified with a plasmid miniprep kit and the plasmid concentration was measured to calculate the copy number. Copy number calculation formula: Copy number (copies / μL) = (plasmid concentration × 10 -9 × 6.02 × 10 23 ) / (660 × number of base pairs).

[0060] Dilute with ddH 2 O to 105 copies / μL, perform 10-fold serial dilutions as the initial concentration, and detect plasmid DNA at different concentrations by RPA / CRISPR-Cas12a fluorescence method and test strip method respectively (according to the experimental method of Example 1) to determine the lowest concentration detectable by RPA / CRISPR-Cas12a, with each concentration gradient repeated 3 times. The experimental results are as shown in the appendix Figure 4 where Figure A is the fluorescence detection intensity result diagram of plasmids with different copy numbers; Figure B is the fluorescence method detection result diagram of plasmids with different copy numbers; Figure C is the test strip method detection result diagram of plasmids with different copy numbers.

[0061] Experimental results: When detected by RPA / CRISPR-Cas12a fluorescence method, obvious fluorescence signals could be detected for plasmid vectors at 10 5 、10 4 、10 3 、10 2 、10 copies / μL (Appendix Figure 4 -A), showing extremely significant differences from the control group. When the plasmid vector was 1 copy / μL, the fluorescence signal value was low. Although there was an extremely significant difference from the control, there was only a weak green light under blue light irradiation (Appendix Figure 4 -B), making it difficult to distinguish, indicating that the detection limit of this method was 10 copies / μL.

[0062] In the RPA / CRISPR-Cas12a test strip detection, the detection results of plasmid vectors at 10 5 、10 4 、10 3 、10 2 、10 copies / μL were positive. When the plasmid concentration was 1 copy / μL, the test strip could not detect it and the result was negative (Appendix Figure 4 -C). It shows that the sensitivity of this system was 10 copies / μL, corresponding to a DNA concentration of 37 ag / μL, and this system had high sensitivity.

[0063] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. In addition, it should be understood that although this specification is described according to the implementation manners, it does not only include one technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other implementation manners that can be understood by those skilled in the art.

Claims

1. A composition for detecting sunflower stem canker pathogen, characterized in that: The composition includes a specific primer RPA-F / R nucleic acid sequence, a specific crRNA nucleic acid sequence and probes FL-ssDNA and LF-ssDNA; The nucleic acid sequence of the primer RPA-F is: 5´-ACATCATGATAATCGTTGGCGCGCCACTGC-3´, SEQ ID NO: 1; The nucleic acid sequence of the primer RPA-R is: 5´-TACTGGTGCAGGAGTGCGGTAGGTGCATAA-3´, SEQ ID NO: 2; The specific crRNA nucleic acid sequence is: 5´-UAAUUUCUACUAAGUGUAGAUGUGUAGGAGAUAAGCGCCUGAU-3´, SEQ ID NO: 3; The probe FL-ssDNA sequence is: 5´-FAM-TTTATTT-BHQ1-3´, the 5´ end of the probe is labeled with a fluorescent group FAM, and the 3´ end is labeled with a quenching group BHQ1; The probe LF-ssDNA sequence is: 5´-FAM-TTTTTTTATTTTTTT-Biotin-3´, the 5´ end of the probe is labeled with a fluorescent group FAM, and the 3´ end is labeled with a quenching group Biotin.

2. A method for detecting sunflower stem canker pathogen based on RPA / CRISPR-Cas12a, characterized in that: The following steps are involved: S1 Design a detection composition: Design a composition for detecting sunflower stem canker pathogen, the composition comprising the specific primer RPA-F / R nucleic acid sequence, the specific crRNA nucleic acid sequence and the probes FL-ssDNA and LF-ssDNA as described in claim 1; S2 DNA extraction: Extract genomic DNA from diseased sunflower tissues to be tested; S3 DNA isothermal amplification: using the genomic DNA obtained in step S1 as a template, adding specific primers RPA-F1 / R1 thereto to perform RPA amplification to obtain an amplified product; S4 CRISPR / Cas12a reaction: The amplified product and crRNA obtained in S2 are mixed with FL-ssDNA and LF-ssDNA probes for cutting reaction to obtain reaction product FL and reaction product LF; S5 Visual Detection: The reaction products FL and LF were placed in a fluorescent PCR instrument and on a Cas12-specific test strip for detection, respectively.

3. A method for detecting sunflower stem canker pathogen based on RPA / CRISPR-Cas12a according to claim 2, characterized in that: The step S1 comprises the following steps: selecting the translation elongation factor of sunflower stem canker bacteria ( EF-1α ) gene partial sequence is the object of primer design, and the specific primer RPA-F / R nucleic acid sequence that meets the RPA reaction conditions is designed. The crRNA nucleic acid sequence is designed within the RPA primer amplification fragment, and the FL-ssDNA and LF-ssDNA probe sequences are designed according to the Cas12a protease recognition site TTTN.

4. A method for detecting sunflower stem canker pathogen based on RPA / CRISPR-Cas12a according to claim 2, characterized in that: The step S2 comprises the following steps: placing the diseased tissue to be detected into ddH2O, mixing thoroughly, and incubating at 100°C for 10 minutes, then using a DNA extraction kit to extract DNA from the diseased tissue, and quantifying the concentration of the extracted DNA using a spectrophotometer.

5. A method for detecting sunflower stem canker pathogen based on RPA / CRISPR-Cas12a according to claim 2, characterized in that: The step S3 includes the following reaction system: 29.4 μL reaction buffer, 2 μL 10 μmol / L upstream primer RPA-F, 2 μL 10 μmol / L downstream primer RPA-R, 5 μL DNA template, and finally 2.5 μL magnesium acetate solution is added, and the rest is supplemented with DEPC-H2O.

6. A method for detecting sunflower stem canker pathogen based on RPA / CRISPR-Cas12a according to claim 5, characterized in that: The isothermal amplification reaction condition in step S3 is a constant temperature incubation at 38° C. for 30 min.

7. A method for detecting sunflower stem canker pathogen based on RPA / CRISPR-Cas12a according to claim 2, characterized in that: The step S4 CRISPR / Cas12a reaction includes the following system: DEPC-H2O 12.5μL, 20U / μL RNase inhibitor 1μL, 10×Cas12a Buffer 2μL, 10μmol / L LbCas12a 1μL, 10μmol / L crRNA 1μL, 10μmol / L FL-ssDNA Reporter or LF-ssDNA Reporter 1μL, and RPA amplification product 2μL.

8. A method for detecting sunflower stem canker pathogen based on RPA / CRISPR-Cas12a according to claim 2, characterized in that: The step S5 comprises the following steps: S51 fluorescence detection: put the reaction product FL into a real-time fluorescence PCR instrument at 38℃ for 30 minutes. After the reaction, put the product under a blue light. If the product does not emit light, the result is negative. If the product emits green fluorescence, the result is positive. S52 test strip test: incubate the reaction system in S4 at 38°C for 20 minutes to obtain the reaction product LF, dilute the reaction product LF 10 times and mix well, immerse the absorption pad end of the Cas12 special test strip into the diluted reaction product LF, and observe whether the quality control line and the test line of the test strip appear; if only one quality control line appears, the reaction result is negative, if the quality control line and the test line appear at the same time or the test line appears alone, the reaction result is positive.

Images