Method for detecting sunflower white rust 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 white rust of sunflower based on RPA / CRISPR-Cas12a, which comprises the following steps: S1, designing a detection composition: designing a composition for detecting white rust of sunflower, 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 detection technology, and by designing a specific primer, crRNA and a probe, the detection duration of the sunflower white rust bacteria is shortened, the detection limit 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 Albugo tragopogi on sunflowers based on RPA / CRISPR-Cas12a. Background Art

[0002] Sunflower (Helianthus annuus.L) is an annual herbaceous plant of the genus Helianthus in the family Asteraceae. It originated in North America, is tolerant to barrenness and salinity, and is an internationally recognized oil crop with excellent quality. It not only has important economic value, but also has ornamental value and medicinal value. In China, sunflower is a characteristic oil crop in the Northeast, Northwest and North China regions, and its cultivation area ranks second only to soybeans and rapeseeds. In recent years, with the increasing consumer demand, the sown area of sunflowers in China has been continuously expanding, making it one of the important oil crops in China.

[0003] To meet the needs of variety adjustment and market supply, China imports a certain amount of oil crops from abroad every year, mainly soybeans and rapeseeds. Imported sunflower seeds have alleviated the domestic market demand for edible oil and feed raw materials to a certain extent. However, with the growth of China's import volume, the risk of the introduction of harmful organisms carried by imported sunflower seeds has increased sharply. The quarantine pests on sunflowers mainly include: Albugo tragopogi, Albugo tragopogi and Phoma macdonaldii, all of which have been listed in the Catalogue of Quarantine Pests for Imported Plants in China. These diseases have not occurred or have occurred locally in China. Once exotic diseases are introduced and spread along with seed transportation, they will have a serious impact on the growth and yield of sunflowers in China. Therefore, the situation of early warning and prevention and control at ports and in the fields is severe.

[0004] Sunflower white rust (Albugo tragopogi (Persoon) var. helianthi Novotelnova) is one of the quarantine diseases that harm sunflowers. It was introduced into China with imported sunflower seeds. Currently, this disease only occurs in the Yili River Valley in Xinjiang, China. This disease can cause damage throughout the growth period of sunflowers, mainly infecting the leaves, stems, petioles and calyces of sunflowers. When the leaf infection is severe, the lesions can connect into patches, causing the leaves to turn yellow and wither, which has a great impact on the yield. After the stems are damaged in the early stage, they become swollen, showing a dark black water-soaked state. In the later stage, the swollen part of the diseased stem loses water, and white powdery sporangia are produced in the depressions. In severe cases, the plants can even lodge. Sunflower white rust not only harms sunflowers, but also harms plants such as chrysanthemums. After the disease occurs, due to the destruction of assimilation, even the severely affected plots may have no harvest. The size, weight and oil content of the seeds infected with sunflower white rust are significantly reduced, and the hull rate increases, which seriously hinders the further improvement of the yield and quality of sunflowers.

[0005] As the first technical defense line for guarding the national biosafety at the border, the customs faces difficulties in the on-site discovery and identification of quarantine diseases in imported products during the process of entry quarantine law enforcement. Harmful organisms such as insects and weeds in sunflower seeds are relatively large in shape and are easy to be discovered and detected on-site. However, after sunflower seeds are harvested in large quantities and go through processes such as sun drying, the damage characteristics of diseases are not obvious at all. After entering the laboratory for detection, the detection cycle is very long, and it is very easy to cause missed detections. At present, in the daily inspections at ports, the quarantine and identification of sunflower white rust are carried out in accordance with the national standard of the People's Republic of China, "Quarantine and Identification Method for Albugo tragopogonis (GB / T 31808-2015)". The detection of the disease identification method adopts techniques such as isolation and culture, ordinary PCR, and fluorescence PCR detection. Among them, methods such as sub-inspection, selecting diseased tissues for isolation and culture, morphological observation, nucleic acid extraction, amplification using corresponding primers, and sequencing are used 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 co-infection of other pathogens, it is difficult to isolate and purify the pathogen, and the time required for identification is long; the detection effect of ordinary PCR detection on seed samples with a low pathogen-carrying rate is not ideal, and false negatives are likely to occur; real-time fluorescence quantitative PCR has the advantages of being not easily contaminated, simple to operate, and high in sensitivity, but it requires special instruments and takes a long time. These commonly used methods must be operated by professional personnel in the laboratory, which cannot meet the efficient supervision efficiency and customs clearance efficiency of the customs and cannot be used for on-site detection. There is an urgent need to develop a cheaper, sensitive, specific, and simple detection method that is more suitable for on-site efficient customs clearance for sunflower white rust. Summary of the Invention

[0006] In view of the problems of long time consumption and easy occurrence of false negatives existing in the current process of customs entry quarantine for Albugo tragopogonis, the present invention provides a method for detecting Albugo tragopogonis based on RPA / CRISPR-Cas12a. This method is based on the RPA / CRISPR-Cas12a detection technology. By designing specific primers and crRNA, the detection accuracy of Albugo tragopogonis is greatly improved, and the detection duration is reduced. The specific technical solution is as follows:

[0007] First of all, the present invention provides a method for detecting Albugo tragopogonis based on RPA / CRISPR-Cas12a, including the following steps:

[0008] S1 Design a detection composition: Design a composition for detecting Albugo tragopogonis, and the composition includes the nucleic acid sequences of specific primers RPA-F / R, the nucleic acid sequence of specific crRNA, and probes FL-ssDNA and LF-ssDNA;

[0009] S2 Extract DNA: Extract the genomic DNA of the sunflower diseased tissue to be detected;

[0010] S3 DNA isothermal amplification: Using the genomic DNA obtained in step S1 as a template, add specific primers RPA-F1 / R1 thereto for RPA amplification to obtain an amplification product;

[0011] S4 CRISPR / Cas12a reaction: Mix and cut the amplification product obtained in S2 and crRNA with FL-ssDNA and LF-ssDNA probes respectively to obtain reaction product FL and reaction product LF;

[0012] S5 Visual detection: Place reaction product FL and reaction product LF in a fluorescence PCR instrument and on a Cas12 special detection test strip for detection respectively.

[0013] Furthermore, the step S1 includes the following steps: Select a partial sequence of the large subunit ribosomal RNA gene of sunflower white rust as the primer design object, design the nucleic acid sequence of specific primers RPA-F / R that meet the RPA reaction conditions and are within the RPA primer amplification fragment, design the crRNA nucleic acid sequence, and design the FL-ssDNA and LF-ssDNA probe sequences according to the Cas12a protease recognition site TTTN.

[0014] Furthermore, the 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 under a spectrophotometer.

[0015] Furthermore, the reaction in the 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 rest is made up with DEPC-H 2 O.

[0016] Furthermore, the isothermal amplification reaction condition in the step S3 is to incubate at 38 °C for 30 min.

[0017] Furthermore, the step S4 CRISPR / Cas12a reaction includes the following system: DEPC-H 212.5 μL of O1, 1 μL of 20 U / μL RNase inhibitor, 2 μL of 10×Cas12a Buffer, 1 μL of 10 μmol / L LbCas12a, 1 μL of 10 μmol / L crRNA, 1 μL of 10 μmol / L FL-ssDNA Reporter or LF-ssDNA Reporter, and 2 μL of RPA amplification product.

[0018] Further, the step S5 includes the following steps:

[0019] 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.

[0020] 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 10 times and mix well. Immerse the absorbent pad end of the Cas12 special test strip into the diluted reaction product LF, and observe whether the quality control line and the detection line appear on the test strip. 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, the reaction result is positive.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The present invention has established a method for detecting Albugo tragopogonis f. sp. helianthi based on RPA / CRISPR-Cas12a. By using the self-designed specific primer sequence and the crRNA nucleic acid sequence with high shearing efficiency, it can specifically detect Albugo tragopogonis f. sp. helianthi, improving the sensitivity of detecting trace amounts of Albugo tragopogonis f. sp. helianthi. The detection sensitivity can reach 10 - 3 copy / μL in the fluorescence method. At the same time, the detection method of the present invention is efficient and rapid. It only takes 80 min from sample preparation to obtaining the detection result, realizing the rapid and sensitive molecular detection of Albugo tragopogonis f. sp. helianthi and meeting the current rapid customs clearance requirements at ports.

[0023] 2) Compared with the prior detection technologies that rely on large and expensive detection instruments such as nucleic acid amplification instruments, the method for detecting Albugo tragopogonis f. sp. helianthi based on RPA / CRISPR-Cas12a established by the present invention only requires a simple test strip and a blue light irradiation lamp to observe the detection result. The operation process is simple and the result is intuitive. At the same time, the reaction technology provided by the present invention has relatively low requirements for the reaction environment and can quickly react at 37 - 42°C, providing convenient conditions for rapid detection and further improving the applicability of the detection method. Description of the Drawings

[0024] Figure 1 This is the experimental result diagram in Experimental Example 1 of a method for detecting Albugo tragopogonis in sunflowers based on RPA / CRISPR-Cas12a of the present invention;

[0025] Figure 2 This is the experimental result diagram in Experimental Example 2 of a method for detecting Albugo tragopogonis in sunflowers based on RPA / CRISPR-Cas12a of the present invention;

[0026] Figure 3 This is the experimental result diagram in Experimental Example 3 of a method for detecting Albugo tragopogonis in sunflowers based on RPA / CRISPR-Cas12a of the present invention;

[0027] Figure 4 This is the experimental result diagram in Experimental Example 4 of a method for detecting Albugo tragopogonis in sunflowers based on RPA / CRISPR-Cas12a of the present invention. Detailed Embodiments

[0028] 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.

[0029] Embodiment 1

[0030] This embodiment provides a method for detecting Albugo tragopogonis in sunflowers based on RPA / CRISPR-Cas12a, including the following steps:

[0031] S1 Design a detection composition: Design a composition for detecting Albugo tragopogonis in sunflowers, which includes, for example, the specific primer RPA-F / R nucleic acid sequence, the specific crRNA nucleic acid sequence, and the probes FL-ssDNA and LF-ssDNA described in Claim 1;

[0032] Specifically, in the NCBI database (National Center for Biotechnology Information), the published gene sequences of Albugo tragopogonis were screened, and a partial sequence of the Large Subunit Ribosomal RNA gene (Genbank: HQ622624.1) was selected as the target for primer design, and specific primers RPA-F / R that meet the RPA reaction conditions were designed. Within the RPA primer amplification fragment, the crRNA nucleic acid sequence was designed. 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.

[0033] Table 1 Sequences of RPA primers, crRNA, and single-stranded reporter molecules

[0034]

[0035] S2 DNA extraction: Extract the genomic DNA of the diseased sunflower tissue to be detected;

[0036] Specifically, to meet the actual detection requirements, a rapid extraction method was used to prepare DNA. The sample with Albugo tragopogonis bacteria was placed in 300 μL of ddH 2 O, thoroughly mixed, incubated at 100 °C for 10 min, and then the supernatant was collected as the crude extracted DNA for rapid detection. The DNA in the supernatant was extracted using the Plant Genomic DNA Kit plant genomic DNA extraction kit. After extraction, the concentration of the extracted DNA was quantified using a spectrophotometer. The extracted DNA was stored at -20 °C for subsequent use.

[0037] S3 Isothermal DNA amplification: Using the genomic DNA obtained in step S1 as a template, specific primers RPA-F1 / R1 were added thereto for RPA amplification to obtain an amplification product;

[0038] Specifically, for the RPA amplification, refer to the instructions of the AmpFuture DNA isothermal rapid amplification kit [specify its specific model]. 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.

[0039] S4 CRISPR / Cas12a reaction: Mix and cut the amplification product obtained in S2 and crRNA with FL-ssDNA and LF-ssDNA probes respectively to obtain reaction product FL and reaction product LF;

[0040] Specifically, the CRISPR / Cas12a reaction system library has the following components: DEPC-H 2 O 12.5 μL, 20 U / μL RNaseinhibitor 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 adding all components, mix well and incubate at 38°C.

[0041] S5 Visual detection: Place reaction product FL and reaction product LF in a fluorescence PCR instrument and on a Cas12 special detection test strip for detection respectively.

[0042] In the present invention, the visual detection includes fluorescence PCR detection and Cas12 special detection test strip detection. During the 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, place the product under a blue light. If the product shows no luminescence, the result is negative. If the product emits green fluorescence, the result is positive.

[0043] 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, the reaction result is positive.

[0044] Experimental Example 1: Specificity Experiment of crRNA Nucleic Acid Sequence

[0045] 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 respectively added to the RPA / CRISPR-Cas12a fluorescence method system, 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:

[0046] 5′-UAAUUUCUACUAAGUGUAGAUCGCUGCAUUCCCAAACAACGCGA-3′, SEQ ID NO: 4,

[0047] The nucleic acid sequence of crRNA2 is:

[0048] 5′-UAAUUUCUACUAAGUGUAGAUGAUGGAAUUUACCACCUGCUUUG-3′, SEQ ID NO: 5,

[0049] crRNA3 is the crRNA in Example 1, and Control is the blank control.

[0050] The experimental results are as shown in the appendix Figure 1 As can be seen from the experimental results, 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 20 minutes, which is higher than that of crRNA1 and crRNA2, indicating that crRNA has a high cleavage efficiency in the system and is suitable for the reaction system of the present invention.

[0051] Experimental Example 2: Specificity Verification of RPA / CRISPR-Cas12a Detection System

[0052] 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 subjected to RPA / CRISPR-Cas12a detection, and the detection results are as shown in the appendix Figure 2 As shown. In the appendix Figure 2 Figure A in it is the specificity verification by fluorescence method; Figure B is the specificity verification by test strip method; in Figures A and B, Treatments 1-3 are strains carrying sunflower white rust, Treatments 4-7 are control strains carrying the corresponding pathogens in Table 2, and Treatment 8 is the blank control.

[0053] Table 2 Information of Test Strains

[0054]

[0055]

[0056] Analysis of experimental results: In the fluorescence detection method (Appendix Figure 2 -A), only 3 strains carrying sunflower white rust showed visible green fluorescence under blue light irradiation, and no luminescence was observed in other tested strains. In the test strip detection method (Appendix Figure 2 -B), the detection results of all 3 strains carrying sunflower white rust were positive, and the detection results of other tested strains were negative. The detection results were consistent with those of the fluorescence method, indicating that this system is specific for the detection of sunflower white rust.

[0057] Experimental Example 3: Detection experiment of simulated samples

[0058] Experimental method: According to the experimental method of Example 1, prepare sporangia with concentrations of 5×10 4 , 5×10 3 , 5×10 2 , 5×10, and 5 per microliter, and simulate the experiment of sunflower seeds carrying bacteria and test it in combination with the RPA-CRISPR / Cas12a detection system for sunflower white rust bacteria. The detection results are shown in Appendix Figure 3 . 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 detection result of real-time fluorescence quantitative PCR for simulated samples. The number of spores per microliter is marked on Figure A.

[0059] Experimental results: The detection results of the RPA / CRISPR-Cas12a fluorescence method and the test strip method ([[]] Figure 3 -A, B) show that the detection results of the samples carrying bacteria are all positive, and the detection results of the sterile sunflower seeds are negative; at the same time, they are consistent with the results of the real-time fluorescence quantitative detection method used in the quarantine and identification method for sunflower white rust bacteria (GB / T 31808-2015) Figure 3 -C), indicating that this system can accurately and quickly detect the pathogen of sunflower white rust.

[0060] Experimental Example 4: Sensitivity experiment

[0061] Experimental method: In the sensitivity determination experiment, plasmid standards 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). Dilute with ddH 2 O to 103 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. Repeat each concentration gradient 3 times. The experimental results are shown in the appendix Figure 4 as follows. 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.

[0062] Experimental results: When detected by RPA / CRISPR-Cas12a fluorescence method, fluorescence signals can be detected for all plasmid vectors (Appendix Figure 4 -A). When the plasmid vector is 10 -3 copy / μL, the fluorescence signal value is low, and only weak green light can be seen under blue light irradiation

[0063] (Appendix Figure 4 -B), which is difficult to distinguish, indicating that the sensitivity of the fluorescence method in this system is 10 -3 copy / μL.

[0064] In the RPA / CRISPR-Cas12a test strip detection, plasmid vectors with 10 3 , 10 2 , 10, and 1 copies / μL are detected as positive. When the plasmid concentration is 10 -2 copy / μL, the test strip cannot detect it, and the result is negative (Appendix Figure 4 -C). It shows that the sensitivity of the test strip method in this system is 1 copies / μL, and this system has high sensitivity.

[0065] 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 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 contain 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 white rust fungus, 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´-CAAGTCAGCACGCAGGATCCGGTCAAGTTC-3´, SEQ ID NO: 1; The nucleic acid sequence of the primer RPA-R is: 5´-CGGTACTTGTTCGCTATCGGTCTCGCACTG-3´, SEQ ID NO: 2; The specific crRNA nucleic acid sequence is: 5´-UAAUUUCUACUAAGUGUAGAUAGUCGCGUUGUUUGGGAAUGCAG-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 white rust based on RPA / CRISPR-Cas12a, characterized in that: The following steps are involved: S1 Design a detection composition: Design a composition for detecting sunflower white rust fungus, 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 white rust based on RPA / CRISPR-Cas12a according to claim 2, characterized in that, The step S1 comprises the following steps: selecting a partial sequence of the large subunit ribosomal RNA gene of sunflower white rust as the primer design object, designing a specific primer RPA-F / R nucleic acid sequence that meets the RPA reaction conditions and within the RPA primer amplification fragment, designing a crRNA nucleic acid sequence, and designing FL-ssDNA and LF-ssDNA probe sequences according to the Cas12a protease recognition site TTTN.

4. A method for detecting sunflower white rust based on RPA / CRISPR-Cas12a according to claim 3, 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 white rust based on RPA / CRISPR-Cas12a according to claim 4, 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 white rust 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 white rust based on RPA / CRISPR-Cas12a according to claim 6, 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, 10 μmol / L FL-ssDNA Reporter or LF-ssDNA Reporter 1μL, and RPA amplification product 2μL.

8. A method for detecting sunflower white rust based on RPA / CRISPR-Cas12a according to claim 7, 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.

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