RPA-CRISPR / Cas12a primer group and probe for visually detecting aphelenchoides besseyi and application of RPA-CRISPR / Cas12a primer group and probe

Abstract

The invention discloses an RPA-CRISPR / Cas12a primer group and a probe for visually detecting aphelenchoides besseyi and application of the RPA-CRISPR / Cas12a primer group and the probe, and belongs to the technical field of molecular detection. A specific primer group and a probe are designed based on a specific sequence on aphelenchoides besseyi genome DNA, and an RPA-CRISPR / Cas12a detection method of aphelenchoides besseyi pathogen is established by combining CRISPR / Cas12a reaction system one-tube detection, and is successfully used for visual detection of existence of aphelenchoides besseyi latent infection in soybean leaves; the detection method is convenient to operate, high in detection speed and suitable for rapidly detecting whether aphelenchoides besseyi with latent infection exists in soybean roots, stems, leaves and bean pods in the field, and visual detection on field infected soybean plants with disease and green diseases is achieved.

Description

Technical Field

[0001] The present invention relates to the field of molecular detection technology, and in particular to an RPA-CRISPR / Cas12a primer set and probe and applications thereof for visually detecting dry-pointed nematodes. Background Art

[0002] In recent years, the main soybean producing areas in the Huanghuaihai area have frequently encountered large-scale outbreaks of soybean "greening disease", resulting in reduced production, and even no harvest in some severely affected areas, which has brought a huge impact on the soybean industry. This disease is caused by the obstruction of seed development, which leads to an imbalance in the "sink-source" relationship of the plant. Infected soybeans remain green when they should be mature and cannot be harvested, and the pods are shriveled or the grains are not full. CALANDRELLI et al. (2023) found that Aphelenchoides besseyi is one of the pathogens of Brazilian soybean greening. The nematode is a migratory internal / external parasitic nematode that can parasitize the roots and aboveground stems, leaves, and pods of soybean plants.

[0003] As soybean plants develop, the dry-tipped nematodes will gradually migrate upward and expand their population. However, plant parasitic nematodes are highly concealed, and early nematode infections are often latent and do not show special symptoms and cannot be identified by the naked eye. Traditional leaf or seedling sampling methods are time-consuming, laborious, and have poor sensitivity, and have many limitations. The development of research on the detection technology system for the dry-tipped nematode, the pathogen of soybean green disease, can detect the latent infection of dry-tipped nematodes in the field in advance. Especially in recent years, with the rapid development of molecular biology, technologies such as PCR have been widely used in the detection of plant parasitic nematodes.

[0004] Piepenburg et al. developed the recombinase polymerase amplification (RPA) technology in 2006. This technology simulates DNA replication in organisms. The core of the technology is that the three enzymes and their mixtures are active at room temperature, and the target product can be amplified under any constant temperature condition between 30°C and 42°C. Because RPA technology has rapid response, strong specificity, high sensitivity, and can be operated on-site in the field, it has been widely used in the diagnosis and detection of plant diseases such as fungi, viruses, and nematodes. The rapid detection technology based on CRISPR / Cas12a has the advantages of high sensitivity and specificity, rapid detection, visual detection, multiple detection capabilities, and simple operation and portability. Combining CRISPR / Cas12a technology with isothermal amplification can achieve rapid nucleic acid amplification and detection. However, RPA-CRISPR / Cas12a rapid detection of the dry tip nematode, the pathogen of soybean disease, has not yet been reported. Summary of the invention

[0005] The purpose of the present invention is to provide an RPA-CRISPR / Cas12a primer set and probe and application for visual detection of dry-tipped nematodes, so as to solve the problems existing in the above-mentioned prior art. The RPA-CRISPR / Cas12a primer set and probe can be used to detect dry-tipped nematodes conveniently and efficiently, and have the advantages of specificity and high sensitivity.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an RPA-CRISPR / Cas12a primer set and probe for visually detecting dry-tipped nematodes, wherein the RPA-CRISPR / Cas12a primer set and probe are:

[0008] RPA18s-F2: 5'-ACACAGATATAATCTACTTGTTCTATGACTTAAA-3';

[0009] RPA18s-R2: 5'-AGTATTATCCAGAGTTAGCAAAAGGACTAA-3';

[0010] Ab-Probe: 5'-AAGACGGGAAGCTGTATGCGTCGAGTCCCA[THF]CAAAAGTACCTTT CT-3'.

[0011] Optionally, the 5' end of the RPA18s-R2 is modified with biotin;

[0012] The 5' end of the Ab-Probe is modified with a fluorescent group, the 30th and 31st positions are modified with THF, and the 3' end is modified with SpacerC3.

[0013] The above-mentioned dry tip nematode is the dry tip nematode that is the pathogen of soybean green disease.

[0014] The present invention also provides a product for visually detecting dry-pointed nematodes, which includes the RPA-CRISPR / Cas12a primer set and probe. The product includes but is not limited to lateral flow chromatography colloidal gold test strips, kits, etc.

[0015] The present invention also provides the use of the RPA-CRISPR / Cas12a primer set and probe in detecting and / or identifying dry-tipped nematodes.

[0016] Optionally, the dry tip nematode includes the dry tip nematode of soybean symptom pathogen.

[0017] The present invention also provides a method for visually detecting dry-pointed nematodes based on RPA-CRISPR / Cas12a, comprising the following steps:

[0018] The genomic DNA of the sample to be tested is used as a template, and the RAP amplification reaction is performed using the RPA-CRISPR / Cas12a primer set and probe. The RAP amplification product is combined with the CRISPR / Cas12a reaction system in a one-tube format for detection, and it is determined whether the sample to be tested is a dry-tipped nematode or contains a dry-tipped nematode according to the detection result. The sample to be tested includes a nematode or a plant sample.

[0019] Optionally, the RAP amplification product is detected in combination with the CRISPR / Cas12a reaction system in one tube, comprising the following steps:

[0020] The RAP amplification product and the sample to be tested are added to the bottom of the reaction tube, and the CRISPR / Cas12a reaction system is dripped onto the inner wall of the reaction tube, and the mixture is shaken to react. If the reaction product shows fluorescence, it indicates that the sample to be tested is a dry-tipped nematode or contains a dry-tipped nematode.

[0021] Optionally, the system of the RPA amplification reaction includes: 31.9 μL of buffer, 22 μL of RPA18s-F, 22 μL of RPA18s-R, 0.6 μL of Ab-Probe, 1 μL of DNA template and 12.5 μL of ddH2O;

[0022] The procedure of the RAP amplification reaction is to react at 37° C. for 5-7 minutes.

[0023] Optionally, the concentration of Cas12a endonuclease in the CRISPR / Cas12a reaction system is 1 μM and the concentration of gRNA is 2 μM.

[0024] Optionally, the dry sharp nematode includes soybean symptomatic nematode pathogenic dry sharp nematode

[0025] In the embodiment of the present invention, in addition to providing the above-mentioned method for visually detecting dry-pointed nematodes based on RPA-CRISPR / Cas12a, ordinary PCR detection and lateral flow chromatography colloidal gold test strip detection are also provided, specifically:

[0026] Ordinary PCR detection: using the genomic DNA of the sample to be tested as a template, using the RPA18s-F2 and RPA18s-R2 to perform PCR amplification reaction, and performing electrophoresis detection on the obtained amplification products. If the amplification products show specific conditions, it indicates that the sample to be tested is a dry-tipped nematode or contains a dry-tipped nematode.

[0027] Lateral flow chromatography colloidal gold test strip detection: Using the genomic DNA of the sample to be tested as a template, the RAP amplification reaction is carried out using the RPA-CRISPR / Cas12a primer set and probe, and the obtained RAP amplification product is detected using a lateral flow chromatography colloidal gold test strip. If the lateral flow chromatography colloidal gold test strip shows both a quality control line and a detection line, it indicates that the sample to be tested is a dry-tipped nematode or contains dry-tipped nematodes; if the lateral flow chromatography colloidal gold test strip only shows a quality control line but no detection line, it indicates that the sample to be tested is not a dry-tipped nematode or does not contain dry-tipped nematodes.

[0028] The present invention discloses the following technical effects:

[0029] (1) High specificity: the primers used specifically target the soybean green nematode pathogen, Acanthopanax spp., and have no specific amplification for the nine related and non-related plant parasitic nematodes tested.

[0030] (2) It has high sensitivity, with the detection limit for the soybean green nematode pathogen, the dry tip nematode, reaching 1 / 1000 of the second-instar larvae.

[0031] (3) The amplification efficiency is high, and the exponential amplification of the target region of soybean green pathogen nematode can be completed in 5 minutes.

[0032] (4) The operation is simple. The RPA amplification reaction can be achieved by using a kit with stable components and mature technology.

[0033] (5) Low requirements for instruments and equipment. 37°C constant temperature amplification only requires handheld reaction tubes, and does not require precise temperature control instruments. When combined with one-tube fluorescence detection, it can also get rid of dependence on various instruments such as gel electrophoresis and ultraviolet imagers.

[0034] (6) It can be detected visually, and the test results can be judged by directly observing the presence or absence of fluorescence with the naked eye.

[0035] In summary, the present invention has higher specificity, sensitivity and convenience than the existing common PCR technology, isothermal amplification technology LAMP technology and RPA-LFD method for detecting dry-tipped nematodes, and the technology can be directly used for the detection of diseased soybeans, which has practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1The figure shows the amplification of dry tip nematodes by RPA primers; M is D2000 marker, 1-36 are 36 dry tip nematode populations collected from different sampling points in Sichuan, Liaoning, Jilin, Jiangsu, Heilongjiang, Anhui and Hebei provinces, and NTC is water;

[0038] Figure 2 It is the optimization diagram of the RPA system; A is the temperature gradient optimization result, and B is the reaction time optimization result;

[0039] Figure 3 It is an optimization diagram of the RPA-CRISPR / Cas12a detection system and incubation method; A is the optimization result of the Cas12a endonuclease concentration gradient; B is the optimization result of the gRNA concentration gradient; C is a one-tube detection diagram of the handheld reaction tube incubation method (1-36 are 36 dry-tipped nematode populations at different collection points, and NTC is sterile water);

[0040] Figure 4 The results of the specific detection experiment of the RPA-CRISPR / Cas12a detection method for dry tip nematodes; A is ordinary PCR electrophoresis detection; B is LFD test strip detection; C is RPA-CRISPR / Cas12a one-tube detection; in AC, 1-36: dry tip nematodes, Hg: soybean cyst nematodes, Ha: wheat cyst nematodes, Hf: Philip cyst nematodes, Hs: beet cyst nematodes, Mg: pseudo-gramineous root-knot nematodes, Me: elephant ear bean root-knot nematodes, Mi: southern root-knot nematodes, Gr: potato golden nematodes, Dd: potato rot stem nematodes; M: D2000 marker;

[0041] Figure 5 The experimental results of using the RPA-CRISPR / Cas12a method established in the present invention to quickly detect dry tip nematodes infecting soybean leaves; 10 2 -10 -4 The corresponding sample concentrations are 100ng / μL, 10ng / μL, 1ng / μL, 0.1ng / μL, 0.01ng / μL, 0.001ng / μL and 0.0001ng / μL respectively. Non-infected refers to samples not infected with dry-pointed nematodes, and NTC is the no-template negative control. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The information of the tested nematodes involved in the following examples is shown in Table 1.

[0048] Table 1 Name, code and source of the tested nematode populations

[0049]

[0050]

[0051] Example 1 Establishment of RPA-CRISPR / Cas12a detection method for dry sharp nematodes

[0052] RPA primers and probes were designed according to the instructions of DNA isothermal rapid amplification kit and using Primer Premier v5.0. The 18S rRNA gene sequences of A. oryzae and closely related nematodes (A. oryzae, A. fragariae and A. ritzemabosi) were searched and compared in NCBI. According to the specific 18S sequence of the genome of A. oryzae (Ab) (GeneBank: MW216945.1), the specific primer pair RPA18s-F2 (5'-ACACAGATATAATCTACTTGTTCTATGACTTAAA-3') / RPA18s-R2 (5'-AGTATTATCCAGAGTTAGCAAAAGGACTAA-3') was designed based on the principle that the RPA amplicon length is 100bp-300bp and the primer length is 30-35bp. The product sequence was analyzed using NCBI, and the fragment with low homology to the downstream primer was selected to design the probe.

[0053] Conventional PCR reaction was used to amplify the primers designed above and detect them by agarose gel electrophoresis. The conventional PCR amplification 25 μL reaction system of dry sharp nematode included: 12.5 μL 2×Rapid Taq Master Mix (purchased from Nanjing Novogene Biotechnology Co., Ltd., No.: P222-02); 0.5 μL of 10 μM forward and reverse primers; 1 μL of template; 8.0 μL of sterile water. The conventional PCR reaction procedure of dry sharp nematode: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15min, 62℃ annealing for 15sec, 72℃ extension for 15sec, a total of 30 cycles; 72℃ extension for 10min; 4℃ storage. The PCR products were separated by electrophoresis on a 2.0% agarose gel stained with GeneRed nucleic acid dye (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., No.: RT211), and photographed and observed using the Tanon Chemi Dog Ultra fully automatic multifunctional imaging system (purchased from Shanghai Tianneng Life Science Co., Ltd.).

[0054] The results of the conventional PCR electrophoresis test of dry sharp nematodes are as follows Figure 1As shown in the figure (in the figure, conventional PCR amplification was performed on 36 dry-tipped nematode populations (i.e., Ab1-Ab36 in Table 1) and water (NTC) collected from different sampling points in Sichuan, Liaoning, Jilin, Jiangsu, Heilongjiang, Anhui, and Hebei provinces; M: D2000 marker), and the electrophoresis detection results showed that the primer pair RPA18s-F2 / R2 could obtain clear and bright electrophoresis bands in all dry-tipped nematode populations, with a band size of 151bp. Based on the fact that the specificity and sensitivity of the RPA reaction are highly dependent on the amplification efficiency of the primers, the screening results showed that the primer pair RPA18s-F2 / R2 is an excellent upstream and downstream primer for the RPA reaction.

[0055] Furthermore, a modified gene tag, i.e., biotin, was added to the 5' end of the downstream primer of the dry-pointed nematode RAP (Ab-RPA). The design of the fluorescent probe is as follows: the amplified product fragment with low homology to the downstream primer sequence is selected to design the probe; between the upstream and downstream primers, a 45 bp sequence that is completely complementary to the target fragment is selected and designed; a FAM fluorescent group tag is added to the 5' end of the sequence, and an abasic site analog (tetrahydrofuran dSpacer, THF) is inserted between the 30th and 31st nucleotides as the recognition site of the nuclease IV (i.e., Nfo enzyme); at the 3' end of the sequence, a C3-Spacer modification group is added to prevent the extension or amplification of the polymerase.

[0056] Furthermore, the gRNA that specifically targets the PAM (TTTA) site of the Cas12a endonuclease was designed based on the location of the specific Ab-RPA primer. The single-stranded DNA reporter probe sequence (FQ1 Reporter) is fluorescein-TTATT-black hole quencher 1. For detailed information on the specific primer pairs, probes, gRNAs, and reporter probe sequences used in the Ab-RPA-CRISPR / Cas12a reaction, see Table 2.

[0057] Table 2 Primers, probes, gRNA and reporter probe sequences designed for RPA-CRISPR / LbaCas12a reaction of dry sharp nematodes

[0058]

[0059] The Ab-RPA primers and probes in Table 2 and the DNA Constant Temperature Rapid Amplification Kit-Basic Type (purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., No.: WLN8203KIT) were used to amplify the dry-pointed nematode-specific genomic fragments.

[0060] The RPA reaction system is: 'buffer A' 29.4μL, 'buffer B' 2.5μL, upstream and downstream primers RPA18s-F2 / R2 (10μM / L) 2μL each, Ab-Probe (10μM) 0.6μL, DNA template 1μL, ddH2O 12.5μL, a total of 50μL. Amplification reaction conditions: 37℃ constant temperature reaction for 5min, 4℃ storage.

[0061] Lateral flow chromatography colloidal gold test strip (LFD test strip) detection reaction: After the RPA reaction is completed, 10 μL of amplification product is fully mixed with 190 μL of sterilized water to dilute the product 20 times, and then tested using lateral flow chromatography colloidal gold test strips (purchased from Hangzhou Ustar Biotechnology Co., Ltd., No.: D003-03), and visually observed after 3-5 minutes. If the test strip shows both the quality control line and the detection line, it indicates that the sample to be tested is dry-tipped nematodes or contains dry-tipped nematodes; if the test strip only shows the quality control line but not the detection line, it indicates that the sample to be tested is not dry-tipped nematodes or does not contain dry-tipped nematodes.

[0062] CRISPR one-tube detection: add 25μL RPA reaction product to the bottom of the reaction tube, add 5μL Cas12a reaction system to the reaction tube (the total volume of the reaction system is 20μL, including LbaCas12a 1μL, 10×NEBuffer2.1reaction buffer 2μL, gRNA0.5μL, reporter probe ssDNA-FQ reporter 1μL and mouse RNase inhibitor 20U). After RPA amplification using optimized reaction conditions, oscillate and mix the RPA reaction product and Cas12a reaction system, and hold the reaction tube to react at 37°C for 5min. Use a portable ultraviolet flashlight to observe, and if there is bright green fluorescence, it indicates that the dry tip nematode DNA is positive.

[0063] Example 2 Optimization of RPA-CRISPR / Cas12a detection system

[0064] On the basis of Example 1, in order to further determine the optimal reaction temperature and reaction time of RPA, the dry sharp nematode genomic DNA (100 ng / μL) was used as a template, and RPA amplification was designed at 4 different reaction temperatures (35, 37, 39 and 42°C); in addition, 6 different time (5, 10, 15, 20, 25 and 30 min) RPA amplification reactions were designed at 37°C. The RPA amplification results are shown in Figure 1. Figure 2As shown in Figure A, the test strip test can simultaneously observe the quality control line and the test line in a wide temperature range of 35°C to 42°C; and there is no significant difference in the brightness of the test line between 35°C and 42°C. In order to facilitate the use of this detection method in the field and at the grassroots level, the optimal reaction temperature of the test line is selected as 37°C. In addition, 6 RPA reactions of different lengths were carried out at 37°C. The results showed that when the reaction time was 5 to 30 minutes, the test line could see the band (see Figure 2 In B); when the reaction time is 5-10 or 25-30 minutes, the detection line band intensity is higher, and both are strong positive bands; while the detection line band intensity obtained after the reaction is carried out for 15-20 minutes is relatively weak, so the optimal reaction time for the detection line is 5-10 minutes.

[0065] In order to further determine the optimal reaction system for the CRISPR / Cas12a detection system, after adding 25 μL of the optimized RPA reaction system to the bottom of the reaction tube, the Cas12a cleavage reaction was designed at 6 different Cas12a endonuclease concentrations (25nM, 50nM, 100nM, 250nM, 500nM and 1μM). In addition, Cas12a cleavage reactions with 5 different gRNA concentrations (250nM, 500nM, 1μM, 2μM and 3μM) were designed at 1μM Cas12a endonuclease concentration, and the fluorescence intensity was detected by qPCR.

[0066] The handheld reaction tube incubation method of the RPA-CRISPR / Cas12a system was validated using 36 dry-pointed nematode populations, and observed using LFD test strips and one-tube detection, respectively.

[0067] CRISPR / Cas12a reaction results Figure 3 As shown, the time it takes for the fluorescence intensity to reach the plateau phase is inversely proportional to the concentration of Cas12a endonuclease, indicating that a high concentration of Cas12a endonuclease concentration can improve the detection sensitivity, so the optimal Cas12a endonuclease concentration is 1 μM. The results of gRNA detection at different concentrations showed that in the concentration range of 250nM-2μM, the time it takes for the fluorescence intensity to reach the plateau phase decreases as the gRNA concentration increases, while when the gRNA concentration is 3μM, the time it takes for the fluorescence intensity to reach the plateau phase increases, indicating that the optimal gRNA concentration is 2μM. In addition, the handheld reaction tubes can efficiently guarantee the RPA reaction and CRISPR / Cas12a cutting reaction, indicating that the handheld reaction tubes meet the detection requirements.

[0068] Example 3 Specific detection test of dry-pointed nematodes using the established RPA-CRISPR / Cas12a detection method

[0069] Thirty-six dry tip nematode populations collected from the field and isolated and purified by the applicant's laboratory were selected, and soybean cyst nematode, wheat cyst nematode, Philip cyst nematode, beet cyst nematode, root knot nematode of gramineous family, root knot nematode of elephant ear bean, southern root knot nematode, potato golden wireworm and potato rot stem nematode were used as control nematode populations (see Table 1). Genomic DNA was extracted from all the tested nematode populations, and the DNA concentration was diluted to 100 ng / μL for conventional PCR amplification and RPA reaction, and the specificity was tested by electrophoresis detection, LFD test strips and CRISPR / Cas12a one-tube detection. Sterile water was used as a no-template control (NTC).

[0070] Specific test results such as Figure 4 As shown, conventional PCR detection was performed using the RPA18s-F2 / R2 primer pair (the reaction system and procedure were the same as in Example 1). The agarose gel electrophoresis results showed that (see Figure 4 In the figure (A), the target product of 151 bp can be specifically amplified for the dry-pointed nematode, while no amplification occurred in the other nine nematodes. This shows that the primer pair RPA18s-F2 / R2 has a high specificity for the dry-pointed nematode.

[0071] In the LFD lateral flow chromatography colloidal gold test strip test process, the RPA amplification reaction of the genomic DNA of 36 dry sharp nematode populations showed double bands of the test line and the quality control line, while other non-target plant parasitic nematodes or no template controls (such as sterile water) only showed the quality control line band, and no test line appeared (see Figure 4 (B).

[0072] The results of the RPA-CRISPR / Cas12a one-tube assay showed that the RPA reaction tubes of the 36 dry-pointed nematode populations all showed green fluorescence, while other non-target plant parasitic nematodes or the no-template control (sterile water) showed no fluorescence (see Figure 4 Middle C).

[0073] Experimental Example 1 Rapid detection of dry-pointed nematodes using the RPA-CRISPR / Cas12a method established by the present invention

[0074] The soybean variety 'Zhonghuang 13' was planted in the greenhouse. After 14 days of planting, the dry sharp nematode suspension was inoculated into the soil, with 1000 second-instar larvae in each pot, and the uninoculated plants were used as negative controls. The roots, stems, leaves and pods of inoculated and uninoculated plants of each variety of soybean were collected, and genomic DNA was extracted using the CTAB method. The initial concentration was 100 ng / μL. After 10-fold gradient continuous dilution, the concentrations were 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL and 0.0001 ng / μL, respectively. It was used as the RPA reaction template for amplification and Cas12a cleavage reaction, and a one-tube fluorescent visualization detection was used. Sterile ddH2O was used as a negative control without template.

[0075] The results are as follows Figure 5 The results showed that the infected soybean leaves could detect strong green fluorescence through the RPA-CRISPR / Cas12a system, and the lower limit of DNA concentration was 10 -3 ng / μL, and there was no significant difference in fluorescence intensity. The test results show that the detection method established by the present invention has high sensitivity to dry-pointed nematodes infecting soybean leaves, can detect them at an early stage, and the genomic DNA of soybean will not affect the test results.

[0076] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An RPA-CRISPR / Cas12a primer set and probe for visual detection of dry-pointed nematodes, characterized in that: The RPA-CRISPR / Cas12a primer set and probe are: RPA18s-F2: 5'-ACACAGATATAATCTACTTGTTCTATGACTTAAA-3'; RPA18s-R2: 5'-AGTATTATCCAGAGTTAGCAAAAGGACTAA-3'; Ab-Probe: 5'-AAGACGGGAAGCTGTATGCGTCGAGTCCCA[THF]CAAAAGTACCTTT CT-3'.

2. The RPA-CRISPR / Cas12a primer set and probe according to claim 1, characterized in that The 5' end of the RPA18s-R2 is modified with biotin; The 5' end of the Ab-Probe is modified with a fluorescent group, the 30th and 31st positions are modified with THF, and the 3' end is modified with SpacerC3.

3. A product for visual detection of dry-pointed nematodes, characterized in that: It includes the RPA-CRISPR / Cas12a primer set and probe described in claim 1 or 2.

4. Use of the RPA-CRISPR / Cas12a primer set and probe as described in claim 1 or 2 in detecting and / or identifying dry-tipped nematodes.

5. The use according to claim 4, characterized in that The dry tip nematodes include the dry tip nematodes caused by soybean symptom.

6. A method for visually detecting dry-pointed nematodes based on RPA-CRISPR / Cas12a, characterized in that: The following steps are involved: Taking the genomic DNA of the sample to be tested as a template, a RAP amplification reaction is performed using the RPA-CRISPR / Cas12a primer set and probe described in claim 1 or 2, and the RAP amplification product is combined with the CRISPR / Cas12a reaction system in a one-tube format for detection, and whether the sample to be tested is a dry-tipped nematode or whether it contains a dry-tipped nematode is determined according to the test result.

7. The method according to claim 6, characterized in that The RAP amplification product is combined with the CRISPR / Cas12a reaction system to detect the one-tube method, which includes the following steps: The RAP amplification product and the sample to be tested are added to the bottom of the reaction tube, and the CRISPR / Cas12a reaction system is dripped onto the inner wall of the reaction tube, and the mixture is shaken to react. If the reaction product shows fluorescence, it indicates that the sample to be tested is a dry-tipped nematode or contains a dry-tipped nematode.

8. The method according to claim 6, characterized in that The system of the RPA amplification reaction includes: 31.9 μL of buffer, 22 μL of RPA18s-F, 22 μL of RPA18s-R, 0.6 μL of Ab-Probe, 1 μL of DNA template and 12.5 μL of ddH2O; The procedure of the RAP amplification reaction is to react at 37° C. for 5-7 minutes.

9. The method according to claim 6, characterized in that The concentration of Cas12a endoprotease in the CRISPR / Cas12a reaction system was 1 μM, and the concentration of gRNA was 2 μM.

10. The method according to claim 6, characterized in that The dry tip nematodes include the dry tip nematodes caused by soybean symptom greening.

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