Rapid fluorescence detection of drug resistance of botrytis cinerea to succinate dehydrogenase bactericides based on RPA-CRISPR / Cas12a

Abstract

The invention discloses a method for detecting drug resistance of botrytis cinerea to succinate dehydrogenase (SDHI) bactericides. On the basis of an RPA-CRISPR / Cas12a system, a specific primer pair amplification target fragment and crRNA for activating Cas12a trans-cleavage activity are designed aiming at key mutation sites related to drug resistance in a bactericide action target SdhB gene, so that fluorescent light visible to naked eyes can be generated only when a resistant strain sequence is matched with the crRNA, and the fluorescent light is opposite to the fluorescent light generated when a sensitive strain sequence is matched with the crRNA. The method provided by the invention overcomes the dependence on laboratories and large instruments in the traditional drug resistance detection method, provides key technical support for accurate prevention and control and drug resistance management of grape gray mold, and provides direct drug use basis for scientific prevention and control in the field.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection of drug resistance in plant pathogenic fungi, specifically relating to an RPA-CRISPR / Cas12a composition and detection method for detecting resistance of Grape Botrytis cinerea to succinate dehydrogenase (SDHI) fungicides. Background Technology

[0002] Grapes are one of my country's important economic fruit trees. With the continuous expansion of the grape industry, diseases have become a significant factor restricting its healthy development. Among these, Botrytis cinerea (Glaucus gracilis) is a major contributing factor. Botrytis cinerea Gray mold, caused by *Botrytis cinerea*, is a key fungal disease that leads to decreased quality and economic losses. *Botrytis cinerea* can damage inflorescences, young fruits, mature fruits, leaves, and fruit stalks. It typically invades host tissues through wounds, natural openings, or by directly penetrating the epidermis. Affected parts show symptoms such as browning, rotting, and sunken areas, and a gray mold layer easily forms under low temperature and high humidity conditions.

[0003] Currently, the control of gray mold in agricultural production mainly relies on chemical agents, including dicarboximides, methoxyacrylates, benzimidazoles, succinate dehydrogenases, pyrroles, phenylaminopyrimidines, and phenylcarbamates. Due to the irrational use of fungicides, rapid genetic variation in the pathogen itself, and the fact that most fungicides have single-site action, gray mold fungi have developed varying degrees of resistance to chemical agents, leading to reduced efficacy. The resistance mechanisms mainly include gene mutations at the drug target site, ectopic excretion via transport, and overexpression of target proteins. Therefore, it is urgent to establish detection techniques for fungicide resistance in gray mold fungi in the field to achieve scientific application of pesticides and efficient control.

[0004] Current methods for detecting antibiotic resistance include traditional phenotypic tests, quantitative ddPCR and qPCR, non-quantitative KASP, LAMP, RFLP, PIRA, and more precise second- and third-generation sequencing technologies. However, traditional phenotypic tests are time-consuming and labor-intensive, and have limitations for unculturable organisms; many of the aforementioned technologies require specialized technicians and complex equipment for resistance detection. Therefore, considering the cost of field application, RPA-CRISPR / Cas12a is more suitable for the immediate detection of antibiotic resistance in pathogens in the field.

[0005] To address the growing challenge of grape botrytis cinerea resistance to succinate dehydrogenase (SDHI) fungicides, this invention combines RPA with CRISPR / Cas12a and utilizes fluorescently labeled probes to construct a rapid fluorescent detection system. This system aims to enable rapid diagnosis of resistance in the field and ultimately contribute to the control of grape botrytis cinerea. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid detection method for grape botrytis cinerea resistance to succinate dehydrogenase (SDHI) fungicides in the field, thereby solving the problems existing in the aforementioned technologies.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A rapid fluorescent detection system based on RPA-CRISPR / Cas12a for detecting resistance of Grape Botrytis cinerea to succinate dehydrogenase (SDHI) fungicides comprises the following components: based on SdhB RPA primer pairs, crRNA, and fluorescently labeled probes for key nucleotide mutation sites in genes.

[0008] The SdhB A CAC mutation to CGC occurs in the gene (SEQ ID No. 5), resulting in the substitution of arginine for histidine at position 272 (Alzohairy, SA). et al . "Cross-resistance of succinate dehydrogenaseinhibitors (SDHI) in Botrytis cinerea and development of molecular diagnostictools for SDHI resistance detection." Phytopathology® 113.6 (2023): 998-1009).

[0009] The gray mold-resistant strain, its SdhB The specific point mutation sequence present in the gene is shown in SEQ ID No. 4 in the sequence listing.

[0010] The amplification includes SdhB In RPA primers for specific point mutations in genes: The upstream primer (BcSdhBF2) nucleic acid sequence is shown in SEQ ID No. 1 of the sequence listing. The nucleic acid sequence of the downstream primer (BcSdhBR1) is shown in SEQ ID No. 2 in the sequence listing.

[0011] To improve the specificity of crRNA recognition, a mismatched base (underlined, U is changed to A) is artificially introduced at mutation site -2; specifically, the nucleic acid sequence of the crRNA (dsSdhB1) used to recognize resistant strains is shown in SEQ ID No. 3 in the sequence listing.

[0012] Only when the resistant strain sequence matches the specific crRNA will the trans-cleavage activity of LbCas12a in the system be activated, indiscriminately cleaving fluorescently labeled probes present in the system, whose 5' end is modified with a FAM fluorescent group and whose 3' end is labeled with a BHQ1 quenching group.

[0013] The nucleic acid sequence of the probe (FQ-Reporter) used to achieve rapid fluorescence detection: 5ˈ6-FAM-TTTAAAAAATTT-BHQ1-3ˈ.

[0014] A further aspect of the present invention provides a rapid fluorescent detection system based on RPA-CRISPR / Cas12a for detecting resistance of Grape Botrytis cinerea to succinate dehydrogenase (SDHI) fungicides, comprising the following steps: S1. Amplify the genomic DNA of the sample to be tested using the RPA primers; S2. After amplification, place the product on ice and prepare the CRISPR-Cas12a reaction system; S3. Add the amplification product to the CRISPR-Cas12a system. After the reaction is complete, irradiate with a 365 nm flashlight and determine whether the sample is a resistant strain based on the green fluorescence.

[0015] Furthermore, in step S1, the genomic DNA of the sample to be tested is extracted using the CTAB method.

[0016] Furthermore, in step S1, the RPA amplification system includes: lyophilized RPA amplification enzyme, RPA reaction buffer (rehydration solution), upstream primer, downstream primer, enzyme-free water (RNase-free water), MgOAc and DNA of the sample to be tested; the amplification conditions are: 35℃, 20 min.

[0017] Furthermore, in step S2, the CRISPR-Cas12a reaction system includes: LbCas12a, a fluorescently labeled probe, NEBuffer 2.1, dithiothreitol, an RNase inhibitor, enzyme-free water, crRNA, and RPA amplification products.

[0018] Furthermore, in step S3, the reaction conditions are: 37°C for 30 min.

[0019] The beneficial effects of this invention are: The technical solution of this invention can solve the problems of time-consuming and labor-intensive traditional phenotypic tests and dependence on large laboratory instruments and equipment. By optimizing the RPA reaction time, reaction temperature, FQ-Reporter concentration, LbCas12a:crRNA ratio, and LbCas12a concentration, the entire detection process of this invention can be completed within 1 hour.

[0020] This invention enables a simple, rapid, and specific fluorescent detection method for detecting resistance to succinate dehydrogenase (SDHI) fungicides in grape gray mold. It provides crucial technical support for the precise control and resistance management of grape gray mold and offers direct guidance for scientific field application of fungicides. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the technical principle of the present invention.

[0022] Figure 2 This is a gel electrophoresis image showing the optimized reaction time of the RPA amplification system in Example 4. M: DL2000 DNA Marker; CK: negative control.

[0023] Figure 3 This is a gel electrophoresis image showing the optimized reaction temperature of the RPA amplification system in Example 4. M: DL2000 DNA Marker; CK: negative control.

[0024] Figure 4 Real-time fluorescence reporting data for the optimized reaction time of the CRISPR-Cas12a detection system in Example 5, CK: negative control.

[0025] Figure 5 The image shows the fluorescence results and signal graphs of the CRISPR-Cas12a detection system FQ-Reporter after final concentration optimization in Example 5. CK: negative control.

[0026] Figure 6 The image shows the fluorescence results and signal diagrams of the optimized LbCas12a:crRNA reaction ratio in the CRISPR-Cas12a detection system in Example 5. CK represents the negative control.

[0027] Figure 7 The image shows the fluorescence results and signal graphs of the optimized final concentration of LbCas12a in the CRISPR-Cas12a detection system in Example 5. CK: negative control.

[0028] Figure 8 The images show the fluorescence results and signal graphs of the CRISPR-Cas12a detection system in Example 6, with CK representing the negative control.

[0029] Figure 9The images show the fluorescence results and signal patterns specific to the CRISPR-Cas12a detection system in Example 7. CK: negative control. Detailed Implementation

[0030] The present invention will be described in detail and completely below through specific embodiments, but this does not limit the invention in any way. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0031] The "resistant strains" mentioned in this invention refer to Botrytis cinerea strains that exhibit resistance to succinate dehydrogenase (SDHI) fungicides. Botrytis cinerea "Sensitive strains" refers to *Botrytis cinerea* strains that are sensitive to succinate dehydrogenase (SDHI) fungicides. Botrytis cinerea ).

[0032] The botrytis strains involved in this invention are all strains preserved by the Institute of Plant Protection, Beijing Academy of Agricultural and Forestry Sciences. Previously, strains that are sensitive to and resistant to succinate dehydrogenase (SDHI) fungicides have been identified using conventional methods.

[0033] First, activated *Botrytis cinerea* strains were inoculated onto PDA plates containing a gradient of boscalid fungicide concentrations (0, 20, 50 μg / mL). After incubation under suitable conditions for 3 to 4 days, the growth status of the strains at each concentration was observed. Strains that grew on the 50 μg / mL drug-containing plate were defined as resistant strains, while those that grew on the lower concentrations were defined as susceptible strains. Genomic DNA was extracted from the activated *Botrytis cinerea* strains using the CTAB method and amplified by conventional PCR. SdhB The gene products were sent to Beijing Nuosai Genome Research Center Co., Ltd. for bidirectional sequencing. The sequencing results were compared and analyzed, and were consistent with the results of the resistance performance test.

[0034] Example 1: Design and screening of specific RPA primers: 1.1 In this invention, botrytis cinerea was selected and screened using conventional methods to obtain a *Botrytis cinerea* strain resistant to succinate dehydrogenase (SDHI) fungicides. The resistant strain was purified and cultured, and its genomic DNA was extracted. Using specific primers IpBcBeg-F and IpBcEnd2-R, the genomic DNA containing... SdhB The target gene at the mutation site was identified. PCR products were sent to Beijing Nuosai Genome Research Center Co., Ltd. for bidirectional sequencing. For sequence alignment analysis, *Botrytis cinerea* strains were downloaded from the NCBI gene database. SdhBThe reference sequence of the gene (accession number: KR866382.1) was used. Multiple sequence alignment analysis was performed between the sequenced data and the reference sequence using MAFFT version 7 online tool, and mutation sites were identified using Bioedit software. Based on the mutation site information, specific RPA primers were designed using Oligo 7.0 software, following the RPA primer design principles in the TwistAmp® DNA Amplification Kit. The designed RPA primers were then sent to Sangon Biotech (Beijing) Co., Ltd. for synthesis.

[0035] Conventional PCR amplification SdhB The primer pair nucleic acid sequence for the gene: IpBcBeg-F: 5ˈ-CCACTCCTCCATAATGGCTGCTCTCCGC-3ˈ (SEQ ID No. 6), IpBcEnd2-R: 5ˈ-CTCATCAAGCCCCCTCATTGATATC-3ˈ (SEQ ID No. 7).

[0036] Standard PCR amplification reaction system (50 μL): 1.1 x S4 Fidelity PCR Mix 44 μL, IpBcBeg-F 2 μL, IpBcEnd2-R 2 μL, template DNA 2 μL.

[0037] Standard PCR amplification procedure: First, incubate at 98℃ for 2 min; then incubate at 98℃ for 10 s, 60℃ for 15 s, and 72℃ for 10 s for 34 cycles; finally, incubate at 72℃ for 5 min; store at 4℃.

[0038] 1.2 RPA primer pair screening To obtain RPA primers with specificity and stable amplification, primer screening was performed according to the RPA reaction system recommended by the manufacturer, TwistAmp® Basic (UK, TwistDX).

[0039] RPA amplification reaction system (50 μL) and procedure: Rehydration buffer 29.5 μL, Forward primer (10 μM) 2.5 μL, Reverse primer (10 μM) 2.5 μL, RNase-free water 11 μL, DNA template 2 μL, MgOAc (280 nM) 2.5 μL.

[0040] Except for MgOAc, all other components were mixed and added to a tube containing lyophilized RPA amplification enzyme. MgOAc was added last to the cap. After centrifugation and mixing, the tube was placed in a 37°C constant temperature instrument for amplification for 30 min.

[0041] 1.3 After the RPA amplification reaction was completed, 40 μL of the amplification product was taken and mixed with 120 μL of ddH2O in a pre-sterilized 1.5 mL centrifuge tube. Then, an equal volume (160 μL) of phenol:chloroform:isoamyl alcohol (25:24:1) was added, vortexed thoroughly, and centrifuged at 12,000 rpm for 5 min. After liquid separation, 10 μL of the supernatant was transferred to a new PCR tube containing 1 μL of 10x DNA loading buffer, vortexed again, and centrifuged at 4000 rpm for 1 min. 6 μL of the product was subjected to 2% agarose gel electrophoresis, using a 2000 bp DNA marker as the molecular weight reference. The reaction conditions were 135 V, 400 mA, and 20 min. The gel after electrophoresis was placed in a gel imaging system to observe the band amplification. The best-performing RPA primer pair (BcSdhBF2 / R1) was selected for subsequent experiments.

[0042] The nucleic acid sequences of the RPA primer pair are shown in SEQ ID No. 1 and SEQ ID No. 2 in the sequence listing.

[0043] SEQ ID No.1:5'-CTCGACATCTTGCCCCTCCTACTGGTG-3'; SEQ ID No. 2: 5'-ACATGTCCTCGAGCAGTTGAGAATAGC-3'.

[0044] Example 2: Design and Screening of crRNA: 2.1 This invention uses resistant strains SdhB Targeted crRNA design was employed at key mutation sites in the gene. To improve the specificity of crRNA recognition and its sensitivity to single nucleotide mutations, the crRNA length was set to 21 bp, ensuring that this length covered the mutation site. To further enhance its ability to distinguish resistant strains, a single-base mismatch was artificially introduced at the -2 position of the crRNA mutation site.

[0045] 2.2 Add the RPA amplification product to the CRISPR-Cas12a system.

[0046] CRISPR-Cas12a reaction system and procedure: (20 μL): DEPC-H2O 12.2 μL, NEBuffer 2.1 (10×) 2 μL, LbCas12a (5 μM) 0.4 μL, RNase inhibitor (40 U / μL) 0.5 μL, DTT (0.1 mmol / L) 0.5 μL, crRNA (10 μM) 0.4 μL, FQ-Reporter (10 μM) 2 μL, RPA amplification product 2 μL.

[0047] The RPA amplification product was added to the PCR tube cap, and after mixing all the reaction components and centrifuging, the tube was placed in a 37°C constant temperature instrument for amplification for 30 min.

[0048] 2.3 Irradiate the CRISPR-Cas12a reaction product with a 365 nm ultraviolet flashlight. If visible green fluorescence appears, the strain is resistant; otherwise, it is susceptible. Finally, the optimal specific crRNA (dsSdhB1) that can distinguish between susceptible and resistant strains is selected.

[0049] The crRNA nucleic acid sequence (dsSdhB1) is shown as SEQ ID No. 3 in the sequence listing.

[0050] SEQ ID No.3:5'-UAAUUUCUACUAAGUGUAGAUuacagaug a cgcacuauucuc-3' .

[0051] Example 3: Design of probes with fluorescent labels Fluorescent probe (FQ-Reporter) nucleic acid sequence: FAM-TTTAAAAAATTT-BHQ1.

[0052] Example 4: Optimization of RPA reaction system To achieve the best amplification effect, the RPA reaction system was optimized in this invention as follows: (1) The RPA reaction temperature was fixed at 37℃, and six time gradients were set at 10, 15, 20, 25, 30, and 35 min. At each preset time point, RPA amplification was performed using both the susceptible strain (strain 175) and the resistant strain (strain 203) of *Botrytis cinerea* as templates, with sterile water as a negative control. The products were analyzed by 2% gel electrophoresis. Figure 2 As shown, the band brightness of the resistant strain tended to stabilize after 20 min. Therefore, 20 min was selected as the optimal RPA amplification time.

[0053] (2) The RPA reaction time was fixed at 20 min, and six temperature gradients were set at 33℃, 35℃, 37℃, 39℃, 41℃, and 43℃. At each preset temperature, RPA amplification was performed using both susceptible and resistant strains of *Botrytis cinerea* (strain 175) as templates, with sterile water used as a negative control. The products were analyzed by 2% gel electrophoresis. Figure 3 As shown, the band brightness of the resistant strain tends to stabilize at 35℃. Therefore, 35℃ is selected as the optimal RPA amplification temperature.

[0054] Example 5: Optimization of CRISPR-Cas12a Detection System To achieve the best detection results, the CRISPR-Cas12a detection system is optimized as follows: (1) With fixed final concentrations of other solutions in the CRISPR-Cas12a reaction system, six time gradients were set at 5, 10, 15, 20, 25, and 30 min. At each preset time point, RPA amplification products of susceptible strain (strain 175) and resistant strain (strain 203) of *Botrytis cinerea* were used, with sterile water as a negative control. The reaction was performed using a real-time quantitative PCR instrument, with fluorescence signal collection frequency set to 1 time / min. After the reaction was terminated, the product was transferred to a new PCR tube, and fluorescence was immediately observed visually under a 365 nm UV flashlight. The experiment was repeated three times. Figure 4 As shown, a significant difference in fluorescence signal intensity was observed between sensitive and resistant strains after 30 minutes of reaction, indicating that this time point can effectively distinguish between sensitive and resistant strains. Therefore, 30 minutes is the optimal detection time for CRISPR-Cas12a.

[0055] (2) Seven fluorescent probe concentrations of 200, 400, 600, 800, 1000, 1200, and 1400 nM were set in the CRISPR-Cas12a reaction system with fixed final concentrations of other solutions. At each preset concentration, RPA amplification products from a susceptible strain of *Botrytis cinerea* (strain 175) and a resistant strain (strain 203) were used, with sterile water as a negative control. The reaction was performed using a real-time quantitative PCR instrument, with fluorescence signal collection frequency set to 1 time / min. After 30 min of reaction, the product was transferred to a new PCR tube, and fluorescence was immediately observed visually under a 365 nm UV flashlight. The experiment was repeated three times. Figure 5 As shown, at the termination of the reaction, a significant difference in fluorescence signal intensity was observed between the sensitive and resistant strains at FQ-Reporter = 1200 nM, indicating that this concentration of fluorescent probe can effectively distinguish between sensitive and resistant strains. Therefore, 1200 nM is the optimal concentration of fluorescent probe.

[0056] (3) The final concentrations of other solutions in the CRISPR-Cas12a reaction system were fixed, and five LbCas12a:crRNA ratios of 2:1, 1:1, 1:2, 1:4, and 1:5 were set. At each preset ratio, RPA amplification products from a susceptible strain of *Botrytis cinerea* (strain 175) and a resistant strain (strain 203) were used, with sterile water as a negative control. The reaction was performed using a real-time quantitative PCR instrument, with fluorescence signal collection frequency set to 1 time / min. After 30 min of reaction, the product was transferred to a new PCR tube, and fluorescence was immediately observed visually under a 365 nm UV flashlight. The experiment was repeated three times. Figure 6 As shown, at the termination of the reaction, the fluorescence signal intensity of the sensitive and resistant strains differed most significantly at an LbCas12a:crRNA ratio of 1:1, indicating that this ratio can effectively distinguish between sensitive and resistant strains. Therefore, 1:1 is the optimal LbCas12a:crRNA ratio.

[0057] (4) Seven LbCas12a concentrations (25, 50, 100, 150, 200, 250, and 300 nM) were set in the CRISPR-Cas12a reaction system to maintain fixed final concentrations. At each preset concentration, RPA amplification products from a susceptible strain (strain 175) and a resistant strain (strain 203) of *Botrytis cinerea* were used, with sterile water as a negative control. The reaction was performed using a real-time quantitative PCR instrument, with fluorescence signal collection frequency set to 1 time / min. After 30 min of reaction, the product was transferred to a new PCR tube, and fluorescence was immediately observed visually under a 365 nm UV flashlight. The experiment was repeated three times. Figure 7 As shown, at the termination of the reaction, the fluorescence signal intensity of the sensitive and resistant strains differed most significantly at LbCas12a = 150 nM, indicating that this concentration can effectively distinguish between sensitive and resistant strains. Therefore, 150 nM is the optimal LbCas12a concentration.

[0058] Example 6: Evaluation of the detection sensitivity of the RPA-CRISPR / Cas12a system To evaluate the detection sensitivity of the RPA-CRISPR / Cas12a system established in this invention, DNA templates from known resistant (strain 203) and susceptible (strain 175) strains were serially diluted 10-fold (43 ng / μL, 4.3 ng / μL, 430 pg / μL, 43 pg / μL, 4.3 pg / μL, 430 fg / μL, 43 fg / μL). At each preset concentration, RPA amplification products from susceptible (strain 175) and resistant (strain 203) strains of *Botrytis cinerea* were analyzed, with sterile water used as a negative control. The reaction was performed using a real-time quantitative PCR instrument, with fluorescence signal collection frequency set to 1 time / min. After 30 min of reaction, the product was transferred to a new PCR tube, and fluorescence was immediately observed visually under a 365 nm UV flashlight. The experiment was performed in triplicate. Figure 8 As shown, at the termination of the reaction, the fluorescence signal was significantly stronger than that of the sensitive strain and the control group when the DNA concentration of the resistant strain was 43 ng / μL, 4.3 ng / μL, and 430 pg / μL. This indicates that the limit of detection of the RPA-CRISPR / Cas12a system established in this invention is 430 pg / μL.

[0059] Example 7: Evaluation of the detection specificity of the RPA-CRISPR / Cas12a system To evaluate the detection specificity of the RPA-CRISPR / Cas12a system established in this invention, the optimized reaction system was used to detect different grape pathogen samples. Primers BcSdhBF2 / R1 were used to detect known grape botrytis cinerea (…). Botrytis cinerea RPA amplification was performed on susceptible (15 strains) and resistant (3 strains) grapes, while simultaneously using a common grape pathogen (6 strains) – *Agropyron cristatum* (the causal agent of grape anthracnose). Colletorichum viniferum and Colletorichum acutatum Grape rot fungus ( Paraeutypella citricola Grape blight pathogen ( Neopestalotiopsis sp. and Cladosporium ) and grape white rot fungus ( Coniella vitis ( ) was used as a non-target control, and sterile water was used as a negative control. The CRISPR-Cas12a reaction was performed in a real-time quantitative PCR instrument, with the fluorescence signal collection frequency set to 1 time / min. After 30 min of reaction, the product was transferred to a new PCR tube, and the fluorescence was immediately observed visually under a 365 nm UV flashlight. The experiment was performed in triplicate. Figure 9 As shown, at the termination of the reaction, significant differences in fluorescence signals were observed between the resistant strains of *Botrytis cinerea*, the susceptible strains, other pathogens, and the control group. This indicates that the RPA-CRISPR / Cas12a system established in this invention has good specificity.

[0060] The above description represents preferred embodiments of the present invention and is used only to illustrate specific implementations of the invention. It is not intended to limit the scope of protection of the invention. Any reasonable modifications, equivalent substitutions, or technical extensions made without departing from the principles of the invention are considered to be within the scope of protection of the invention.

Claims

1. A fluorescent rapid detection system for resistance of Botrytis cinerea to succinate dehydrogenase class fungicides based on RPA-CRISPR / Cas12a, characterized in that, Comprising the following components: based on SdhB RPA primer pairs, crRNA and probes with fluorescent markers designed for key nucleotide mutation sites in genes; the RPA primer pair consists of an upstream primer as shown in SEQ ID No. 1 in the sequence listing and a downstream primer as shown in SEQ ID No. 2 in the sequence listing; the sequence of the crRNA is as shown in SEQ ID No. 3 in the sequence listing; the structure of the fluorescently labeled probe is: 5ˈ6-FAM-TTTAAAAAATTT-BHQ1-3ˈ; FAM is the fluorescent group of the probe, and BHQ1 is the quenching group of the probe.

2. A fluorescence rapid detection method for resistance of Botrytis cinerea to succinate dehydrogenase class fungicides based on RPA-CRISPR / Cas12a, characterized in that, detecting a sample to be detected by using the visual detection composition of claim 1, comprising the following steps: S1. amplifying the genomic DNA of the sample to be detected by using the RPA primer pair; S2. placing the product after amplification on ice, and configuring a CRISPR-Cas12a reaction system; S3. adding the amplification product to the CRISPR-Cas12a system, after the reaction is completed, irradiating with a 365 nm flashlight, and judging whether the sample to be detected is a resistant strain according to the green fluorescence.

3. The method for rapid detection of fluorescence of Botrytis cinerea resistant to succinate dehydrogenase inhibitors according to claim 2, characterized in that, In step S1, the genomic DNA of the sample to be detected is extracted by the CTAB method.

4. The method for rapidly detecting the resistance of Botrytis cinerea to succinate dehydrogenase inhibitors according to claim 2, wherein, In step S1, the RPA amplification system comprises: freeze-dried RPA amplification enzyme, RPA reaction buffer, upstream primer, downstream primer, enzyme-free water, MgOAc and sample DNA to be detected; and the amplification condition is: 35℃, 20 min.

5. The method for rapidly detecting the resistance of Botrytis cinerea to succinate dehydrogenase inhibitors according to claim 2, wherein, In step S2, the CRISPR-Cas12a reaction system comprises: LbCas12a, fluorescently labeled probe, NEBuffer 2.1, dithiothreitol, RNase inhibitor, enzyme-free water, crRNA and RPA amplification product.

6. The method for rapidly detecting the resistance of Botrytis cinerea to succinate dehydrogenase inhibitor fungicides according to claim 2, wherein, In step S3, the reaction condition is: 37℃, 30 min.

7. The method for rapidly detecting the resistance of Botrytis cinerea to succinate dehydrogenase inhibitor fungicides according to claim 2, characterized in that, The concentration of the fluorescently labeled probe in the CRISPR-Cas12a system is 1200 nM.

8. The method for rapidly detecting the resistance of Botrytis cinerea to succinate dehydrogenase inhibitors according to claim 2, wherein, The final concentration of the LbCas12a in the CRISPR-Cas12a system is 150 nM.

9. The method for rapidly detecting the resistance of Botrytis cinerea to succinate dehydrogenase inhibitors according to claim 2, wherein, In the CRISPR-Cas12a system, the molar ratio of LbCas12a:crRNA is 1:1.

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