A method for visual detection of Dactylonectria spp., the causal agent of grape black root rot, based on RPA-CRISPR / Cas12a and its application.

Abstract

The application discloses a method and application for visual detection of grape guignardia based on RPA-CRISPR / Cas12a Dactylonectria The detection method comprises RPA specific amplification primers, crRNA and ssDNA probes. TUB The RPA specific amplification primers take the gene as a specific recognition target, and can be amplified specifically for the guignardia genus. Dactylonectria The method has high specificity and sensitivity, and is simple, fast and convenient in operation process, 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. Dactylonectria spp. The method. 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.macrodidima、D.novozelandica、D.torresensis、Ilyonectria liriodendri、Neonectria.sp.1 The pathogen causing black root rot infects the root system through wounds or soil, leading to blackening and necrosis of the roots, discoloration of the xylem tissue, stunted growth, and even death of the plant. The external symptoms of black root rot are often indistinguishable from other grapevine diseases and resemble those associated with 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 differentiated from other factors is crucial for developing scientific control measures, preventing disease spread, and mitigating industry losses. As the severity of grape black root rot increases, there is an urgent need to research rapid detection systems and design detection methods suitable for field application, establishing 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 detection 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. Dactylonectria Four main pathogenic bacteria were selected. TUB Using the β-tubulin sequence as the detection target, 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, the rapid amplification, recognition, 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. DactylonectriaThe RPA primer pair composition of spp.; capable of amplifying specific gene fragments of the target pathogenic fungus;

[0010] Among them, detection Dactylonectria The primer pairs for spp. are composed of Dactylonectria RPA forward primers for spp. and Dactylonectria The RPA reverse primer composition of spp.;

[0011] Dactylonectria The sequence of the RPA forward primer (TUB-dac-101-130F) for spp. is as follows:

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

[0013] Dactylonectria The RPA reverse primer (TUB-dac-260R) sequence for spp. is as follows:

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

[0015] crRNA can specifically guide Cas12a to recognize and cleave 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] Dactylonectria The crRNA (D-crRNA-7) sequence of spp. is as follows:

[0017] 5'-UAAUUUCUACUAAGUGUAGAUGUGCAAUAUAGGUCCACCUCCAGA-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 detection method for grape black root rot based on RPA-CRISPR / Cas12a visualization mainly includes the following steps:

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

[0022] S2: Use the DNA obtained from 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: Dactylonectria spp . The optimal reaction conditions are 41°C and 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: Dactylonectria The optimal reaction conditions for spp. are 35°C and 20 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. Dactylonectria spp. for D. alcacerensis , D. macrodidyma , D. novozelandica , D. torresensis One or more of them.

[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... Dactylonectria alcacerensis、D. macrodidyma、D. New Zealand, D. of TorresFor 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 41 °C for 30 minutes, followed by a CRISPR / Cas12a reaction at 35 °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 fg / μL level. Detection results can be visualized using lateral flow chromatography test strips. This invention... Dactylonectria The lowest concentration detected was 8 fg / μL. This method requires no complex equipment or specialized operation, enabling early, rapid, and accurate detection of the pathogen of grape black root rot in field conditions, which helps to achieve timely early warning and control of the disease. Attached Figure Description

[0042] Figure 1 For amplification Dactylonectria The RPA primer specificity detection results for spp. are shown in the figure, with lanes M: DL2000 DNA Manker; 1: Cadophora luteo-olive ;2: Apple dorsal bud mold C.malorum ;3: C.sabaouae ;4: Phaeoacremonium iranianum ;5: Paraeutypella citricola ;6: Neocosmospora solani ;7: Sickle cell disease ;8: Fusarium annulatum ;9: Fusarium oxysporum F.oxysporum ;10: Fusarium moniliformes F.proliferatum ;11: Fusarium argentis F. acuminatum ;12: You will be a diaspora. ;13: Soybean interset shells D.soybean ;14: Brown-spotted schizocarp Rosellinia necatrix ;15: White Rot Calycanthophytes Coniella vine ;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 wine-producing ;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 Dactylonectria The crRNA screening results of spp. are shown in the figure. D1-D8 represent D-crRNA-1, D-crRNA-2, D-crRNA-3, D-crRNA-4, D-crRNA-5, D-crRNA-6, D-crRNA-7, and D-crRNA-8, 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... Dactylonectria Figure B shows the optimal temperature for the RPA reaction of spp. DNA. Dactylonectria Optimization diagram of RPA reaction time for spp. DNA.

[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 results. Dactylonectria Figure B shows the optimal reaction temperature for CRISPR / Cas12a in spp.; Figure B is the detection... Dactylonectria Optimized reaction time diagram for CRISPR / Cas12a in spp.

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

[0047] Figure 6 For the detection of CRISPR / Cas12a in this invention Dactylonectria Screening results of cas12a protein concentration in spp.

[0048] Figure 7 For the detection of CRISPR / Cas12a in this invention Dactylonectria Visualization results of spp. specific lateral flow chromatography test strips; 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 Dactylonectria spp. 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 the 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 41℃ 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... DactylonectriaThe optimal RPA primers for spp. are TUB-dac-101-130F / TUB-dac-260R, 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 anthrax

[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 35℃ for 20 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... Dactylonectria The optimal crRNA for spp. is D-crRNA-7, such as... Figure 2 .

[0058] Dactylonectria The optimal primers, crRNA, and reporter ssDNA sequences for spp. are as follows:

[0059] Dactylonectria The sequence of the RPA forward primer (TUB-dac-101-130F) for spp. is as follows:

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

[0061] Dactylonectria The RPA reverse primer (TUB-dac-260R) sequence for spp. is as follows:

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

[0063] Dactylonectria The crRNA (D-crRNA-7) sequence of spp. is as follows:

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

[0065] The sequence of the ssDNA probe (CR-DNA-FB-2) 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. Dactylonectria The optimal reaction temperature for spp. is 41°C. 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 successful amplification and relatively bright bands were observed was selected as the optimal time for the RPA amplification stage of this invention, thus screening out… Dactylonectria The optimal reaction time for spp. is 30 minutes, such as Figure 3 B.

[0069] In summary, amplification was selected. Dactylonectria The optimal reaction conditions for spp. are 41°C and 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: 31, 33, 35, 37, 39, 41, and 43 °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. Dactylonectria The optimal reaction temperature for spp. is 35°C. 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... Dactylonectria The optimal reaction time for spp. is 20 minutes, such as Figure 4 B.

[0073] In summary, the detection methods used in the CRISPR / Cas12a detection and recognition stage were selected. Dactylonectria The optimal reaction conditions for spp. are 35°C and 20 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 final concentration ratio for this stage of the invention, thus screening out the detection... Dactylonectria The optimal ratio of spp. 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... Dactylonectria 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 obtained from 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 Dactylonectria The optimal reaction conditions for spp. are 41°C and 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 Dactylonectria The optimal reaction conditions for spp. are 35°C and 20 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 reaction 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 TUB-dac-101-130F / TUB-dac-260R, 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*. Dactylonectria The test strips for amplified DNA from *S. spp.* 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 Dactylonectria The RPA-CRISPR / Cas12a visualization detection technology for grape black root causal agent spp. is specific.

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

[0100] Grape black root rot fungus Dactylonectria spp . ( Dactylonectria spp. for D.alcacerensis , D.macrodidyma , D.novozelandica , D.torresensis Genomic DNA was serially diluted 10-fold to obtain DNA at concentrations of 80 ng / μL, 8 ng / μL, 800 pg / μL, 80 pg / μL, 8 pg / μL, 800 fg / μL, 80 fg / μL, and 8 fg / μL to determine the sensitivity of the detection technology. The method described in Example 3 was used with primer pair TUB-dac-101-130F / TUB-dac-260R and optimized reaction system and conditions for detection. The results showed that for… Dactylonectria When the DNA concentration of *S. spp.* was 80 ng / μL-80 fg / μL, a clear band appeared on the test strip, but when the DNA concentration was 8 fg / μL, no band appeared on the test strip. Figure 8 .

[0101] The above embodiments all use primer pairs TUB-dac-101-130F / TUB-dac-260R and detection Dactylonectria The reaction system and reaction conditions of spp. 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 detection method for grape black root rot fungus based on RPA-CRISPR / Cas12a Dactylonectria The composition of spp., consisting of an RPA primer pair, crRNA, and an 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; crRNA is the nucleotide fragment shown in SEQ ID No. 3 of 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; Dactylonectria spp. for D. alcacerensis , D.macrodidyma , D.novozelandica , D. torresensis One or more of them.

2. The RPA-CRISPR / Cas12a-based detection method for grape black root rot as described in claim 1 Dactylonectria Application of the composition of spp. in the detection of grape black root rot fungus, said grape black root rot fungus is Dactylonectria spp. caused by infection, the aforementioned Dactylonectria spp. for D. alcacerensis , D.macrodidyma , D.novozelandica , D. torresensis One or more of them.

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