A method and application for visual detection of grape black root causal agent Ilyonectriasp. based on RPA-CRISPR / Cas12a.

Abstract

The application discloses a method and application for visual detection of grape phialophora genus based on RPA-CRISPR / Cas12a. Ilyonectria The method comprises RPA specific amplification primers, CrRNA and ssDNA probes. HIS The RPA specific amplification primers take the gene as a specific recognition target, and can be amplified specifically for the grape phialophora genus. Ilyonectria The method for detecting the grape phialophora genus has high specificity and sensitivity, and the operation process is simple and fast, and is suitable for rapid screening and preliminary diagnosis of the target pathogen in a field environment or a primary detection scene.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology for plant fungal pathogens, specifically relating to a visualization detection method for grape black root rot fungus based on RPA-CRISPR / Cas12a. Ilyonectria sp. methods and applications. Background Technology

[0002] Grape branch diseases are among the most destructive diseases in global grape cultivation, and their impact has been increasing in recent years, seriously threatening the sustainable development of the grape industry. These diseases mainly include five types: grape canker, grape black root rot, grape wilt, grape vine blight, and grape top rot. Caused by various fungi, they affect both root and branch tissues, exhibiting complex pathogenesis and diverse symptoms. The pathogens often have long incubation periods and are highly concealed, frequently showing typical symptoms only after widespread transmission in the field, posing a significant challenge to timely diagnosis and control. Currently, there are no therapeutic treatments; therefore, early identification and rapid detection of these diseases are crucial.

[0003] Of all grapevine diseases, black root disease is considered the most destructive, especially threatening vines under 8 years old, and is particularly damaging to nurseries. There are many types of pathogens; in my country, 5 genera and 10 species of pathogens have been identified as causing black root disease in grapes. Campylocarpon fasciculare, Cylindrocladiella lageniformis, C.peruviana, C.viticola, Dactylonectria alcacerensis, D.macrodidyma, D.novozelandica, D.torresensis, Ilyonectria liriodendri, Neonectria.sp.1 Black root rot pathogens infect the root system through wounds or soil, causing root blackening and necrosis, discoloration of xylem tissue, stunted growth, and even plant death. The external symptoms of black root rot are often indistinguishable from other grapevine diseases and resemble those of abiotic diseases such as spring frost, winter damage, nutrient deficiencies, and water stress. Therefore, early detection of the pathogen before symptoms are obvious or easily distinguishable from other factors is crucial for developing scientific control measures, preventing disease spread, and mitigating industry losses. As black root rot worsens, there is an urgent need to research and design rapid detection systems suitable for field application, and to establish sensitive, accurate, simple, and low-cost early pathogen identification techniques.

[0004] Traditional detection methods include pathogen isolation, morphological observation, and molecular identification, such as PCR, qPCR, nested PCR, and multiplex PCR. While these methods offer high sensitivity and accuracy, they are dependent on laboratory conditions, equipment, and professional personnel, making rapid on-site screening difficult and limiting their application in the field. More importantly, grape black root rot is easily confused with similar symptoms caused by other branch diseases and abiotic factors, making accurate diagnosis difficult based solely on external symptoms.

[0005] With the development of molecular biology techniques, isothermal amplification (RPA) has become an important alternative to traditional PCR, especially recombinase polymerase amplification (RPA) technology, which is particularly suitable for field use due to its rapid reaction (usually within 30 minutes), independence from temperature cycling equipment, and ease of operation. To further improve the specificity and visualization of detection, RPA is often combined with the CRISPR / Cas12a system. Cas12a can activate its cleavage activity after recognizing the target nucleic acid, and output a visual signal in conjunction with fluorescent probes or lateral flow dipsticks, enabling rapid detection without instruments.

[0006] Therefore, constructing an integrated rapid field detection method based on RPA-CRISPR / Cas12a not only combines the rapid amplification capability of RPA with the highly specific recognition capability of Cas12a, but also enables rapid on-site diagnosis of the pathogen of grape black root rot in the early stages of disease when symptoms are not obvious. This method has broad potential for field application. Developing a portable, low-cost, and highly sensitive RPA / CRISPR detection method is expected to become a core tool in the grape black root rot control system, helping to achieve accurate monitoring and early control of the disease, thereby effectively curbing its spread and ensuring the healthy and sustainable development of the grape industry. Summary of the Invention

[0007] This invention aims to develop a rapid detection technology based on isothermal amplification and molecular recognition, constructing a field-based, visualized detection method using a combination of RPA amplification, a CRISPR / Cas12a system, and lateral flow chromatography test strips. This technical solution features a simple operation process and short reaction time, meeting the rapid diagnostic needs of grape black root rot fungus under field conditions and expanding the technical avenues for agricultural pathogen detection at the grassroots level. The method provided by this invention targets [specific pathogens] in grape black root rot fungus. Ilyonectria sp. A major pathogenic bacterium, selected HIS Using histone gene sequences as detection targets, corresponding RPA-specific primer pairs, crRNA sequences, and fluorescently labeled ssDNA reporter probes were designed. Combined with the Cas12a-mediated specific nucleic acid cleavage and signal release mechanism, rapid amplification, identification, and visualization of pathogen nucleic acids were achieved.

[0008] Based on this, the present invention provides a composition for visual detection of grape black root rot fungus based on RPA-CRISPR / Cas12a, comprising RPA primers, crRNA, and ssDNA probes.

[0009] The RPA primers are used for detection. IlyonectriaThe RPA primer pair composition for sp.; capable of amplifying specific gene fragments of the target pathogenic fungus;

[0010] Among them, detection Ilyonectria The primer pairs for sp. are derived from Ilyonectria sp. RPA forward primer and Ilyonectria The RPA reverse primer composition of sp.;

[0011] Ilyonectria The RPA forward primer (HIS-il-95-124F) sequence for sp. is as follows:

[0012] 5'-ACGCGTCTTGCAACATCTATCTTCATCACA-3' (SEQ ID No. 1),

[0013] Ilyonectria The RPA reverse primer (HIS-il-318R) sequence for sp. is as follows:

[0014] 5'-GACCTGTGCCGGGTGTGTTAGAGTGAATGC-3' (SEQ ID No. 2).

[0015] crRNA can specifically guide Cas12a to recognize and cut dsDNA targets with PAM sites or suboptimal PAM sites. After cas12a forms a ternary complex with crRNA and target DNA, it will also be activated to perform non-specific cleavage of ssDNA.

[0016] Ilyonectria The crRNA (I-crRNA-9) sequence of sp. is as follows:

[0017] 5'-UAAUUUCUACUAAGUGUAGAUUCUACCGGUGGUGUCAAGAAGCCU-3' (SEQ ID No. 3).

[0018] The ssDNA probe is a single-stranded DNA with FAM fluorescent and biotin groups modified at both ends, respectively, to generate a visual signal during Cas12a-mediated nonspecific cleavage. The structure of the ssDNA probe (CR-DNA-FB-2) is: 5'-FAM-TTTATTT-Biotin-3'; FAM is the fluorescent group of the probe, and biotin is the biotin group, which binds to the biotin antibody in the lateral flow chromatography test strip, thereby achieving visual detection.

[0019] The composition enables rapid, specific, and visual detection of grape black root pathogens under simple field conditions.

[0020] The RPA-CRISPR / Cas12a-based visual detection method for grape black root rot mainly includes the following steps:

[0021] S1: DNA extraction from the sample to be tested;

[0022] S2: Use the DNA extracted in step S1 using RPA primers to perform RPA amplification;

[0023] S3: Add the product from step S2 into the CRISPR-Cas12a reaction system to perform the CRISPR / Cas12a reaction; in this step, crRNA is used to guide Cas12a to recognize and cleave the RPA amplification product and activate its non-specific cleavage activity.

[0024] S4: Add 30 μL ddH2O to dilute the product and generate a visual signal using the Cas12 / 13 dedicated nucleic acid test strip. Read the result within ten minutes.

[0025] In step S1, the DNA extraction steps include:

[0026] (a) The isolated strains were cultured on potato dextrose (PDA) medium in a climate chamber (25°C) for 7–14 days.

[0027] (b) Collect the sample to be tested and grind it into powder;

[0028] (c) DNA was extracted using the CTAB method.

[0029] (d) DNA quality and concentration were determined using agarose gel electrophoresis and NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA).

[0030] (e) Dilute the gDNA to an appropriate concentration with double-distilled water (ddH2O) and store at -20°C for subsequent experiments.

[0031] In step S2, the RPA reaction system and reaction procedure are as follows:

[0032] Reaction system: 29.5 μL Rehydration buffer, 2.5 μL each of 10 μM RPA forward primer and 10 μM RPA reverse primer, 11 μL RNase-free water, 2 μL DNA template; after mixing, quickly transfer to a reaction tube containing amplification reaction enzyme powder, and finally add 2.5 μL MgOAc (280 nM) to the cap of the reaction tube, and centrifuge to mix.

[0033] Reaction conditions: Ilyonectria The optimal reaction conditions for sp. are 39°C for 30 minutes.

[0034] In step S3, the CRISPR / Cas12a reaction system and reaction procedure are as follows:

[0035] Reaction system: 12.2 μL NEBuffer 2.1, 0.4 μL LbCas12a (5 μM), 0.5 μL RNase inhibitor (40 U / μL), 0.5 μL DTT (0.1 mM), 2 μL ssDNA probe (10 μM), 0.4 μL crRNA (10 μM), 2 μL RPA amplification product.

[0036] Reaction conditions: Ilyonectria The optimal reaction conditions for sp. are 39°C for 15 minutes.

[0037] In step S4, the detection method of the Cas12 / 13 dedicated nucleic acid test strip (such as a lateral flow chromatography test strip) is as follows:

[0038] After the CRISPR / Cas12a reaction is complete, add 30 μL of sterile ultrapure water to the reaction system. Insert the conjugate pad end of the Cas12 / 13 nucleic acid test strip into the centrifuge tube. The control line will begin to develop color after about 1-2 minutes. The test result is valid if the test strip is read within 10 minutes. According to the color development of the test strip, if both the control line and the test line appear, it is a positive sample; if only the control line appears, it is a negative sample.

[0039] In the method, the sample to be tested is selected from one or more of grapevines, grape seedlings, and grape rhizosphere soil samples. Ilyonectria sp. for I.liriodendri .

[0040] The beneficial effects of this invention are:

[0041] This invention establishes a rapid, on-site, visualized detection technology for the pathogen of grape black root rot, based on recombinase polymerase amplification (RPA) and the CRISPR / Cas12a system. Specifically... Ilyonectria liriodendriFor pathogenic bacteria, RPA-specific primers and corresponding crRNAs were designed at the genus level, and key parameters in the reaction system were optimized. The method involves RPA amplification at approximately 39°C for 30 minutes, followed by a CRISPR / Cas12a reaction at 39°C, achieving detection signal output within 15–20 minutes. By optimizing the system conditions, the detection method of this invention exhibits high sensitivity and specificity, capable of detecting nucleic acids down to the pg / μL level. Detection results can be visualized using lateral flow chromatography test strips. This invention... Ilyonectria The lowest concentration of sp detected was 3.6 pg / μL. This method requires no complex equipment or professional operation and can achieve early, rapid, and accurate detection of grape black root rot pathogens in a field environment, which helps to realize timely early warning and control of the disease. Attached Figure Description

[0042] Figure 1 For amplification Ilyonectria The RPA primer specificity detection results for sp. are shown in the figure, with lanes M: DL2000 DNA Manker; 1: Cadophora luteo-olivacea ;2: Apple dorsal bud mold C.malorum ;3: C.sabaouae ;4: Phaeoacremonium iranianum ;5: Paraeutypella citricola ;6: Neocosmospora solani ;7: N.falciformis ;8: Fusarium annulatum ;9: Fusarium oxysporum F.oxysporum ;10: Fusarium moniliformes F.proliferatum ;11: Fusarium argentis F.acuminatum ;12: Diaportha eres ;13: Soybean interset shells D.sojae ;14: Brown-spotted schizocarp Rosellinia necatrix ;15: White Rot Calycanthophytes Coniella vitis ;16: Cylindrocladiella lageniformis ;17: C.peruviana ;18: C.viticola ;19: Dactylonectria alcacerensis ;20: D.macrodidyma ;twenty one: D.novozelandica ;twenty two: D.torresensis ;twenty three: Ilyonectria liriodendri ;24: Staphylococcus aureus Botryosphaeria dothidea ;25: Dioscorea opposita Lasiodiplodia theobromae ;26: Neodiscrystosporium Neopestalotiopsis sp.;27: Cladosporium Cladosporium ;28: Colletotrichum viniferum ;29: Anthrax fusiformis C.acutatum ;30: Fusarium anthracis C.falcatum;31:CK - ;32:CK + CK - Negative control (ddH2O), CK + Positive control: The positive control reagent included in the RPA kit.

[0043] Figure 2 For testing Ilyonectria The results of crRNA screening for sp. are shown in the figure. I1-I10 represent I-crRNA-1, I-crRNA-2, I-crRNA-3, I-crRNA-4, I-crRNA-5, I-crRNA-6, I-crRNA-7, I-crRNA-8, I-crRNA-9, and I-crRNA-10, respectively.

[0044] Figure 3 This is an agarose gel electrophoresis image showing the optimized RPA amplification conditions in this invention. M: DL2000 DNA Marker. Negative controls were all performed using ddH2O. Image A shows the amplification... Ilyonectria Figure B shows the optimal RPA reaction temperature for sp., and Figure B shows the amplification... Ilyonectria Optimization diagram of RPA reaction time for sp.

[0045] Figure 4 This is a visualization of the CRISPR / Cas12a-optimized lateral flow chromatography test strip of the present invention. Negative controls were all achieved using ddH2O. C represents the control line, and T represents the detection line. Figure A shows the detection... Ilyonectria Figure B shows the optimal reaction temperature for CRISPR / Cas12a sp.; Figure B is the detection... Ilyonectria Optimized reaction time diagram of sp. CRISPR / Cas12a.

[0046] Figure 5 For the detection of CRISPR / Cas12a in this invention Ilyonectria Screening results of the ratio of crRNA to Cas12a protein concentration of sp.

[0047] Figure 6 For the detection of CRISPR / Cas12a in this invention Ilyonectria Figure showing the screening results of cas12a protein concentration in sp.

[0048] Figure 7 For the detection of CRISPR / Cas12a in this invention Ilyonectria Visualization of the results of a specific lateral flow chromatography test strip; M: DL2000 DNA Manker; 1: Cadophora luteo-olivacea ;2: Apple dorsal bud mold C.malorum ;3: C.sabaouae ;4: Phaeoacremonium iranianum ;5: Paraeutypella citricola ;6: Neocosmospora solani ;7: N. falciformis ;8: Fusarium annulatum ;9: Fusarium oxysporum F. oxysporum ;10: Fusarium moniliformes F. proliferatum ;11: Fusarium argentis F. acuminatum ;12: Diaportha eres ;13: Soybean interset shells D.sojae ;14: Brown-spotted schizocarp Rosellinia necatrix ;15: White Rot Calycanthophytes Coniella vitis ;16: Cylindrocladiella lageniformis ;17: C. peruviana ;18: C.viticola ;19: Dactylonectria alcacerensis ;20: D.macrodidyma ;twenty one: D.novozelandica ;twenty two: D. torresensis ;twenty three: Ilyonectria liriodendri ;24: Staphylococcus aureus Botryosphaeria dothidea ;25: Dioscorea opposita Lasiodiplodia theobromae ;26: Neodiscrystosporium Neopestalotiopsis sp.;27: Cladosporium Cladosporium ;28: Colletotrichum viniferum ;29: Anthrax fusiformis C. acutatum ;30: Fusarium anthracis C. falcatum ;31:CK - ;32:CK + CK - : Negative control (ddH2O).

[0049] Figure 8 For the detection of CRISPR / Cas12a in this invention Ilyonectria sp. sensitivity results graph. Detailed Implementation

[0050] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0051] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0052] Example 1: Design and screening of RPA primers and crRNA

[0053] This invention research involved reviewing literature and searching and downloading information on the pathogens of grape black root disease and other grape diseases from the NCBI website. Tub and His Gene sequence. Based on the MAFFT-aligned sequence, the specific bases of the target pathogen DNA were identified. Using primer design software Oligo 7.0, following the design principles of RPA primers: primer length 30-35 bp, amplified fragment length 100-200 bp. Specific bases were placed at the 3' end of the primers to ensure specificity. After determining the optimal primers with the highest specificity, a PAM site was selected based on the RPA amplified fragment. The standard PAM site is 5'-TTTN-3', but if a standard PAM site is unavailable, suboptimal PAM sites such as 5'-VTTV-3', 5'-TTVV-3', 5'-TTNT-3', 5'-TVTV-3', or 5'-TTVV-3' can be selected. This invention uses a suboptimal PAM site. A nucleic acid fragment approximately 20-25 bp after the PAM site was used as the crRNA spacer sequence. Simultaneously, an ssDNA strip labeled with 6-CarboxyFurtherin (6-FAM) at one end and biotin as a quenching group at the other end was designed as a reporter molecule. The reporter molecule is a non-specific cleavage target activated after the formation of a ternary complex between crRNA, cas12a protein, and target DNA. It can bind to a lateral flow chromatography test strip, making the detection results visible.

[0054] Primer screening was performed using RPA amplification. Specifically, the 50 μL amplification system consisted of: 29.5 μL rehydration buffer, 2.4 μL each of 10 μM forward and reverse primers, 11.2 μL RNase-free water, and 2 μL DNA template. After mixing, the mixture was quickly transferred to a reaction tube containing amplification enzyme powder. Finally, 2.5 μL of MgOAc (280 nM) was added to the cap of the reaction tube, and the mixture was centrifuged. The reaction conditions were 39℃ for 30 min. After amplification, 150 μL of ddH2O was added for dilution, followed by purification and extraction with an equal volume of a 25:24:1 phenol-chloroform-isoamyl alcohol solution. The amplified product was then detected by 1.5% agarose gel electrophoresis, and the results were observed using gel imaging. Primers that amplified bands using *Gnaphalium affine* DNA as a template while other pathogens did not amplify were selected. To verify the specificity of the primers, pathogens of other grape diseases were used as controls. Specific strain information is shown in Table 1. All strains shown in Table 1 were identified using conventional methods. Amplification... Ilyonectria The optimal RPA primers for sp. are HIS-il-95-124F / HIS-il-318R, such as Figure 1 .

[0055] Table 1.

[0056] strain number Diseases Types of pathogens 1 Grape wilt disease 2 Grape wilt disease Apple dorsal bud mold 3 Grape wilt disease 4 Grape wilt disease 5 Grape rot 6 Grape sickle root rot 7 Grape sickle root rot 8 Grape sickle root rot 9 Grape sickle root rot Fusarium oxysporum 10 Grape sickle root rot Fusarium moniliforme 11 Grape sickle root rot Fusarium argentis 12 Grapevine blight 13 Grapevine blight Soybean interset shell 14 Grape white spot disease Brown-spotted sclerotium 15 Grape white rot White rot spores 16 Grape black root disease 17 Grape black root disease 18 Grape black root disease 19 Grape black root disease 20 Grape black root disease 21 Grape black root disease 22 Grape black root disease 23 Grape black root disease 24 grape canker Staphylococcus aureus 25 grape canker Cocoyoma dispora 26 Grape vine blight Neodiscus polychaete. 27 Grape vine blight Cladosporium 28 Grape anthracnose 29 Grape anthracnose Anthrax bacillus 30 Grape anthracnose Fusarium anthracis

[0057] The optimal RPA primers were used to screen crRNAs in the CRISPR / Cas12a reaction stage. The 20 μL reaction system contained: 12.2 μL NEBuffer 2.1, 0.4 μL LbCas12a (5 μM), 0.5 μL RNase inhibitor (40 U / μL), 0.5 μL DTT (0.1 mM), 2 μL CR-DNA-FB-2 (10 μM), 0.4 μL crRNA (10 μM), and 2 μL RPA amplification product. The reaction conditions were 39℃ for 15 min. After the reaction, 30 μL of ddH2O was added. The binding pad end of the Cas12 / 13 nucleic acid detection test strip was inserted into a centrifuge tube. The control line began to develop color after approximately 1-2 minutes. Based on the color development of the test strip, crRNAs showing both the control and test lines for *Gnaphalium affine*, while other pathogens only showed the control line, were screened. The detection... Ilyonectria The optimal crRNA for sp. is I-crRNA-9, such as Figure 2 .

[0058] Ilyonectria The optimal primers, crRNA, and reporter ssDNA sequence for sp. are as follows:

[0059] Ilyonectria The RPA forward primer (HIS-il-95-124F) sequence for sp. is as follows:

[0060] 5'-ACGCGTCTTGCAACATCTATCTTCATCACA-3' (SEQ ID No. 1),

[0061] Ilyonectria The RPA reverse primer (HIS-il-318R) sequence for sp. is as follows:

[0062] 5'-GACCTGTGCCGGGTGTGTTAGAGTGAATGC-3' (SEQ ID No. 2).

[0063] Ilyonectria The crRNA (I-crRNA-9) sequence of sp. is as follows:

[0064] 5'-UAAUUUCUACUAAGUGUAGAUUCUACCGGUGGUGUCAAGAAGCCU-3' (SEQ ID No. 3).

[0065] The ssDNA probe (CR-DNA-FB-2) sequence is: 5'-FAM-TTTATTT-Biotin-3'. FAM is a fluorescent group, and Biotin is a biotin group.

[0066] Example 2: Optimization of RPA Detection Technology System

[0067] (1) In the RPA amplification stage of this invention, the reaction time was initially set to 35 minutes, and seven temperature gradients (33, 35, 37, 39, 41, 43, and 45) were set sequentially. A negative control was set at each temperature. Based on the results of agarose gel electrophoresis, the temperature at which successful amplification and relatively bright bands were selected was set as the optimal temperature for the RPA amplification stage of this invention, and amplification was screened. Ilyonectria The optimal reaction temperature for sp. is 39℃, such as Figure 3 A.

[0068] (2) In the RPA amplification stage of this invention, using the previously screened optimal reaction temperature, eight reaction times were sequentially set: 5, 10, 15, 20, 25, 30, 35, and 40 degrees Celsius. A negative control was set at each time. Based on the results of agarose gel electrophoresis, the temperature at which amplification was successful and the band was relatively bright was selected as the optimal time for the RPA amplification stage of this invention, and the amplification was screened. Ilyonectria The optimal reaction time for sp. is 30 minutes, such as Figure 3 B.

[0069] In summary, amplification was selected. Ilyonectria The optimal reaction conditions for sp. are 39°C for 30 minutes.

[0070] Example 3: Optimization of CRISPR / Cas12a detection technology system

[0071] (1) In the CRISPR / Cas12a detection and identification stage of this invention, the reaction time was first set to 35 minutes, and the reaction temperature was optimized by setting seven temperature gradients: 33, 35, 37, 39, 41, 43, and 45 °C. A negative control was set for each reaction temperature. Based on the results of the lateral flow chromatography test strips, the temperature at which the strips were successfully identified and cut, and which showed relatively clear bands, was selected as the optimal temperature for this stage of the invention. Ilyonectria The optimal reaction temperature for sp. is 39℃, such as Figure 4 A.

[0072] (2) In the CRISPR / Cas12a detection and identification stage of this invention, the reaction time is optimized based on the previously screened optimal temperature. Seven time gradients are set sequentially: 5, 10, 15, 20, 25, 30, 35, and 40 minutes. A negative control is set for each reaction time. Based on the results of the lateral flow chromatography test strip, the temperature at which successful identification and cutting of the strip with relatively clear bands is selected as the optimal reaction time for this stage of the invention, thus screening out the detection... Ilyonectria The optimal reaction time for sp. is 15 minutes, such as Figure 4 B.

[0073] In summary, the detection methods used in the CRISPR / Cas12a detection and recognition stage were selected. Ilyonectria The optimal reaction conditions for sp. are 39°C for 15 minutes.

[0074] (3) In the CRISPR / Cas12a detection and identification stage of this invention, the final concentrations of crRNA and Cas12a protein in the reaction system were optimized based on the previously screened optimal reaction conditions. Seven different final concentration ratios were set sequentially: 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1. A negative control was set for each different ratio. Based on the results of the lateral flow chromatography test strips, the ratio that was successfully identified and cut and had relatively clear bands was selected as the optimal concentration ratio for this stage of the invention, thus screening out the detection... Ilyonectria The optimal ratio of crRNA to Cas12a protein in the system is 2:1, such as... Figure 5 .

[0075] (4) In the CRISPR / Cas12a detection and recognition stage of this invention, the optimal reaction conditions and the concentration ratio of crRNA to Cas12a protein have been screened. Based on the screened conditions, the optimal concentration of Cas12a protein in the system is determined. Eight different protein concentrations (25, 50, 75, 100, 125, 150, 175, and 200 nM) are set sequentially, with a negative control for each concentration. Considering the results displayed by the lateral flow chromatography test strip, subsequent storage, and operation method, the concentration that was successfully identified and cut, and showed relatively clear bands, is selected as the optimal Cas12a protein concentration for this stage of the invention, thus screening out the detection... Ilyonectria The optimal concentration of Cas12a protein in the system is 100 nM, such as... Figure 6 .

[0076] Through experiments in Examples 1-3, it was finally determined that the RPA-CRISPR / Cas12a-based visual detection method for grape black root rot, as described in this invention, mainly includes the following steps:

[0077] S1: DNA extraction from the sample to be tested;

[0078] S2: Use the DNA extracted in step S1 using RPA primers to perform RPA amplification;

[0079] S3: Add the product from step S2 into the CRISPR-Cas12a reaction system to perform the CRISPR / Cas12a reaction; in this step, crRNA is used to guide Cas12a to recognize and cleave the RPA amplification product and activate its non-specific cleavage activity.

[0080] S4: Add 30 μL ddH2O to dilute the product and generate a visual signal using the Cas12 / 13 dedicated nucleic acid test strip. Read the result within ten minutes.

[0081] In step S1, the DNA extraction steps include:

[0082] (a) The isolated strains were cultured on potato dextrose (PDA) medium in a climate chamber (25°C) for 7–14 days.

[0083] (b) Collect the sample to be tested and grind it into powder;

[0084] (c) DNA was extracted using the CTAB method.

[0085] (d) DNA quality and concentration were determined using agarose gel electrophoresis and NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA).

[0086] (e) Dilute the gDNA to an appropriate concentration with double-distilled water (ddH2O) and store at -20°C for subsequent experiments.

[0087] In step S2, the RPA reaction system and reaction procedure are as follows:

[0088] Reaction system: 29.5 μL Rehydration buffer, 2.5 μL each of 10 μM RPA forward primer and 10 μM RPA reverse primer, 11 μL RNase-free water, 2 μL DNA template; after mixing, quickly transfer to a reaction tube containing amplification reaction enzyme powder, and finally add 2.5 μL MgOAc (280 nM) to the cap of the reaction tube, and centrifuge to mix.

[0089] Reaction conditions:

[0090] Detection Ilyonectria The optimal reaction conditions for sp. are 39°C for 30 minutes.

[0091] In step S3, the CRISPR / Cas12a reaction system and reaction procedure are as follows:

[0092] Reaction system: 12.2 μL NEBuffer 2.1, 0.4 μL LbCas12a (5 μM), 0.5 μL RNase inhibitor (40 U / μL), 0.5 μL DTT (0.1 mM), 2 μL ssDNA probe (10 μM), 0.4 μL crRNA (10 μM), 2 μL RPA amplification product.

[0093] Reaction conditions:

[0094] Detection Ilyonectria The optimal reaction conditions for sp. are 39°C for 15 minutes.

[0095] In step S4, the detection method of the Cas12 / 13 dedicated nucleic acid test strip (such as a lateral flow chromatography test strip) is as follows:

[0096] After the CRISPR / Cas12a reaction is complete, add 30 μL of sterile ultrapure water to the reaction system. Insert the conjugate pad end of the Cas12 / 13 nucleic acid test strip into the centrifuge tube. The control line will begin to develop color after about 1-2 minutes. The test result is valid if the test strip is read within 10 minutes. According to the color development of the test strip, if both the control line and the test line appear, it is a positive sample; if only the control line appears, it is a negative sample.

[0097] Example 4: Specificity evaluation of RPA-CRISPR / Cas12a detection technology

[0098] Combining the optimized RPA amplification and CRISPR recognition conditions, with sterile water as a negative control, the DNA of the 30 test strains mentioned in Table 1 of Example 1 was amplified using the primer pair HIS-il-95-124F / HIS-il-318R, as described in Example 3, and then detected using CRISPR / Cas12a test strips. The results showed that the 30 strains were infected with *Gnaphalium affine*. Ilyonectria The test strips for the amplified DNA products of *Sp.* showed distinct red bands at both the control and test lines, while the amplified products using DNA from other grape pathogens and the negative control as templates only showed red bands at the control line. Figure 7 The results show that for Ilyonectria The visualization technique for detecting grape black root causal agent sp. using RPA-CRISPR / Cas12a is specific.

[0099] Example 5: Sensitivity Evaluation of RPA-CRISPR / Cas12a Detection Technology

[0100] Grape black root rot fungus Ilyonectria sp. ( I.liriodendri Genomic DNA was serially diluted 10-fold to obtain concentrations of 360 ng / μL, 36 ng / μL, 3.6 ng / μL, 360 pg / μL, 36 pg / μL, and 3.6 pg / μL. The sensitivity of the detection technique was determined using these serially diluted DNA samples. The method described in Example 3 was used with primer pair HIS-il-95-124F / HIS-il-318R, along with optimized reaction systems and conditions, for detection. The results showed that... Ilyonectria When the DNA concentration of *Sp. sp.* was 360 ng / μL-3.6 ng / μL, a clear band appeared on the test strip. However, when the DNA concentration was 360 pg / μL, no band appeared on the test strip. Figure 8 .

[0101] The above embodiments all used primer pairs HIS-il-95-124F / HIS-il-318R and used detection... Ilyonectria sp . The reaction system and reaction conditions were tested.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit it. Various modifications and changes can be made without departing from its scope.

Claims

1. A visual detection method for grape black root rot fungus based on RPA-CRISPR / Cas12a Ilyonectria The composition of sp., consisting of RPA primer pairs, crRNA and ssDNA probe, is characterized by, Among them, R The PA primer pair consists of the RPA forward primer shown in SEQ ID No. 1 of the sequence listing and the RPA reverse primer shown in SEQ ID No. 2 of the sequence listing; Among them, crRNA is the nucleotide fragment shown in SEQ ID No. 3 in the sequence listing; The structure of the ssDNA probe is: 5'-TTTATTT-3'; The ssDNA probe has a fluorescent group attached to its 5' end and a biotin group attached to its 3' end; the fluorescent group is FAM or HEX.

2. A visual detection method for grape black root rot fungus based on RPA-CRISPR / Cas12a, characterized in that, The composition for visual detection of grape black root rot as described in claim 1 is used to detect the presence of the pathogen in the test sample. Ilyonectria liriodendri Infection detection steps include: S1: DNA extraction from the sample to be tested; S2: RPA amplification reaction of the DNA obtained in step S1 is performed using RPA primers; S3: Add the product from step S2 into the CRISPR-Cas12a reaction system to carry out the CRISPR / Cas12a reaction; S4: Add 30 μL ddH2O to dilute the product and generate a visual signal using the Cas12 / 13 dedicated nucleic acid test strip. Read the result within ten minutes.

3. The method according to claim 2, characterized in that, In step S1, the DNA extraction of the sample to be tested includes the following steps: (a) The isolated strains were cultured on potato dextrose medium in an artificial climate chamber at 25°C for 7–14 days. (b) Collect samples of the bacterial strain to be tested and grind them into powder; (c) DNA was extracted using the CTAB method; (d) DNA quality and concentration were determined using agarose gel electrophoresis and NanoDrop 2000; (e) Dilute the gDNA to an appropriate concentration with double-distilled water and store at -20°C for subsequent experiments.

4. The detection method according to claim 2, characterized in that, In step S2, the total volume of the reaction system used for RPA amplification is 50 μL. The RPA amplification reaction system is prepared as follows: 29.5 μL Rehydration buffer, 2.5 μL each of 10 μM RPA forward primer and 10 μM RPA reverse primer, 11 μL RNase-free water, and 2 μL DNA template; after mixing, quickly transfer to a reaction tube containing amplification enzyme powder, and finally add 2.5 μL of 280 nM MgOAc to the cap of the reaction tube, and centrifuge to mix. The RPA amplification reaction conditions are as follows: Detection Ilyonectrialiriodendri The reaction conditions are 39°C for 30 minutes.

5. The detection method according to claim 2, characterized in that, In step S3, the total volume of the CRISPR / Cas12a reaction system is 20 μL, and the reaction system consists of: 12.2 μL NEBuffer 2.1, 0.4 μL 5 μL MbCas12a, 0.5 μL 40 U / μL RNase inhibitor, 0.5 μL 0.1 mM DTT, 2 μL 10 μM ssDNA probe, 0.4 μL 10 μM CrRNA, and 2 μL RPA amplification product. The CRISPR / Cas12a reaction conditions are as follows: Detection Ilyonectria liriodendri The reaction conditions are 39°C for 15 minutes.

6. The method according to claim 2, characterized in that, In step S4, the detection method of the Cas12 / 13 dedicated nucleic acid test strip is as follows: After the CRISPR / Cas12a reaction in step S3 is completed, add 30 μL of sterile ultrapure water to the reaction system. Insert the conjugate pad end of the Cas12 / 13 nucleic acid test strip into the centrifuge tube. The control line will start to develop color after 1-2 minutes. The test result is valid if the test strip is read within 10 minutes. According to the color development of the test strip, if both the control line and the test line appear, it is a positive sample; if only the control line appears, it is a negative sample.

7. The method according to claim 6, wherein the Cas12 / 13 dedicated nucleic acid detection test strip is a lateral flow chromatography test strip.

8. The method according to claim 2, characterized in that, The samples to be tested are selected from one or more of the following: grapevines, grape seedlings, and grape rhizosphere soil samples.

9. The RPA-CRISPR / Cas12a-based visual detection method for grape black root rot as described in claim 1 Ilyonectrialiriodendri The application of the composition in the detection of grape black root rot fungus, wherein the grape black root rot fungus is Ilyonectrialiriodendri Caused by infection.

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