Primer group for tomato yellow mottle related virus detection based on RT-RAA-CRISPR / Cas12a and visualization method

Abstract

The invention provides a primer group for detecting a tomato yellow mottle related virus TYMaV based on RT-RAA-CRISPR / Cas12a. The primer group comprises an RT-RAA amplification primer pair and a CRISPR / Cas12a detection probe. The invention provides a method for visually and rapidly detecting TYMaV. The method comprises the following steps: extracting total RNA (Ribonucleic Acid) of tobacco leaves infected by TYMaV or coarsely extracted virions; the method comprises the following steps: preparing RT-RAA reaction mixed liquid at the bottom of a PCR tube and CRISPR premixed liquid at the cover of the PCR tube, adding total RNA or crude extraction virus particles into the RT-RAA reaction mixed liquid, incubating in a 42 DEG C metal bath for 30 minutes, placing the CRISPR premixed liquid at the bottom of the tube through centrifugation or shaking, mixing with an RT-RAA reaction product, incubating in a 37 DEG C metal bath for 20 minutes, adding 80 mu L of ddH2O, and detecting a target strip by using a flow measurement chromatography test strip. The method can be used for preparing a kit for detecting TYMaV. According to the primer group and the established TYMaV visual rapid detection method based on RT-RAA-CRISPR / Cas12a, a theoretical basis and a technical guarantee are provided for monitoring, predicting, forecasting, preventing and treating the TYMaV in the field.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease detection, and particularly relates to a primer set for detecting tomato yellow mottle-associated virus (TYMaV) based on RT-RAA-CRISPR / Cas12a, and a method and application for its visual rapid detection. Background Art

[0002] Tomato yellow mottle-associated virus (TYMaV) is a cytoplasmic rhabdovirus that can infect tomato ( Solanum lycopersicum L.) crops, and was first discovered in Chongqing, China in 2017. Its genome is a negative-sense single-stranded RNA (Xu et al., 2017). The virus was successively discovered on pepper and black nightshade in China in 2022. Our research group first detected the virus on tobacco collected from the southwestern Guizhou region of China in 2022 (Huang et al., 2024). Through sampling and detection, black nightshade in the southwestern Guizhou region also carries TYMaV, which indicates the risk of TYMaV spreading among solanaceous crops.

[0003] Currently, common methods for detecting plant RNA viruses mainly include serological detection, reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), reverse transcription recombinase polymerase amplification technology (RT-RPA), etc. Serological detection is an effective method for routine detection, but it requires specific antibodies, and the process of producing antibodies is cumbersome and time-consuming. The detection results of RT-PCR and RT-LAMP methods established using molecular biology are faster and more sensitive, but the cost of the detection equipment and reagents required is relatively high, which cannot meet the needs of rapid field detection.

[0004] Reverse transcription recombinase-aided amplification (RT-RAA) is a new type of isothermal amplification technology. After reverse transcription, it can amplify template DNA sensitively, specifically, and rapidly. The combination of RT-RAA and lateral flow strip (LFS) technology based on CRISPR / Cas12a can achieve the visual detection of plant RNA viruses in the field, providing a basis and technical guarantee for the rapid detection of plant viruses. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a primer set for detecting Tomato yellow mottle-associated virus TYMaV based on RT-RAA-CRISPR / Cas12a, and a method and application for its visual rapid detection, aiming at the deficiencies of the above-mentioned existing technologies. This method uses the RNA of field-collected samples or virus particles roughly extracted with PBS buffer as a template, adds it to the RT-RAA reaction solution containing specific primers at the bottom of the PCR tube, reacts for 30 min under the constant temperature condition of 42 °C, and then mixes it with the CRISPR premix containing Cas12a protein, crRNA and probe in the PCR tube cap. After reacting at 37 °C for 20 min and combining with a flow-through chromatographic test strip, the detection result can be obtained. The operation is simple and the detection time is shortened. The reaction equipment is simple, and a metal bath can meet the conditions. The whole reaction is completed in a single PCR tube, reducing the probability of cross-contamination. The detection sensitivity is 10 times higher than that of RT-PCR. By adding a recognition probe to the reaction, the specificity of the detection method is ensured. Through the flow-through chromatographic test strip, the visualization of the detection result is realized. Based on the above advantages, the TYMaV visual rapid detection method based on RT-RAA-CRISPR / Cas12a established by the present invention provides a theoretical basis and technical guarantee for the monitoring, prediction and prevention of TYMaV in the field.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a primer set for detecting Tomato yellow mottle-associated virus based on RT-RAA-CRISPR / Cas12a, including an RT-RAA amplification primer pair and a CRISPR / Cas12a detection probe; The RT-RAA amplification primer pair includes primer TYMaV-F and primer TYMaV-R; The nucleotide sequence of the primer TYMaV-F is as shown in SEQ ID NO:1; The nucleotide sequence of the primer TYMaV-R is as shown in SEQ ID NO:2; The CRISPR / Cas12a detection probe is TYMaV-crRNA, and the nucleotide sequence is as shown in SEQ ID NO:3.

[0007] The present invention also provides a method for the visual rapid detection of Tomato yellow mottle-associated virus using the above-mentioned primer set for detecting Tomato yellow mottle-associated virus based on RT-RAA-CRISPR / Cas12a. This method is as follows: S1. Extract the total RNA of tobacco leaves infected with Tomato yellow mottle-associated virus TYMaV to obtain the total RNA; Or roughly extract the virus particles of tobacco leaves infected with Tomato yellow mottle-associated virus TYMaV with PBS buffer to obtain the roughly extracted virus particles; S2. Configure the RT-RAA reaction mixture: Add the recombinant enzyme freeze-dried powder to the RT-RAA reaction tube, and then add 25 μL of RAA buffer, 16 μL of ddH2O, 2.4 μL of primer TYMaV-F, 2.4 μL of primer TYMaV-R, 3 μL of 280 mM magnesium acetate solution, and 4 μL of probe TYMaV-LF. Dissolve the enzyme to obtain the RT-RAA reaction mixture. The sequence of the probe TYMaV-LF is Biotin-TTTTTT-FAM. S3. Configure the CRISPR premix: The system of the CRISPR premix is as follows: 3 μL of 10×NEBuffer3.1, 1 μL of LbCas12a, 2 μL of TYMaV-crRNA, 1 μL of dithiothreitol, and 1 μL of RNA inhibitor. S4. Add the RT-RAA reaction mixture obtained in S2 to the bottom of the PCR tube, then add 1 μL of the total RNA or the crude virus particles obtained in S1. Then, place the CRISPR premix obtained in S3 inside the PCR tube cap. Incubate in a metal bath at 42 °C for 30 min, and the RT-RAA reaction product is obtained at the bottom of the tube. Place the CRISPR premix inside the tube cap at the bottom of the tube by centrifugation or shaking, mix it with the RT-RAA reaction product at the bottom of the tube, incubate in a metal bath at 37 °C for 20 min, then add 80 μL of ddH2O, and detect the target band with a flow-through chromatographic test strip. If both the test line and the control line show red bands, the sample is positive; if only the control line shows a red band, the sample is negative; if the control line does not show a band, the test strip is invalid.

[0008] The present invention also provides the application of the above-mentioned primer set for detecting tomato yellow mottle-associated virus based on RT-RAA-CRISPR / Cas12a. The primer set for detecting tomato yellow mottle-associated virus based on RT-RAA-CRISPR / Cas12a is used to prepare a kit for detecting tomato yellow mottle-associated virus TYMaV.

[0009] The present invention has the following advantages compared with the prior art: The present invention uses the RNA of field-collected samples or crudely extracted virus particles as a template, adds it to the RT-RAA reaction solution containing specific primers at the bottom of a PCR tube, reacts for 30 min under the constant temperature condition of 42 °C, and then mixes it with the CRISPR premix containing Cas12a protein, crRNA and probe on the lid of the PCR tube. After reacting at 37 °C for 20 min, the detection result can be obtained. The operation is simple and the detection time is shortened. The reaction equipment is simple, and a metal bath can meet the conditions. The whole reaction is completed in a single PCR tube, reducing the probability of cross-contamination. The detection sensitivity is 10 times higher than that of RT-PCR. By adding a recognition probe in the reaction, the specificity of the detection method is ensured. Through a flow-through chromatography test strip, the visualization of the detection result is realized. The visual rapid detection method of TYMaV based on RT-RAA-CRISPR / Cas12a established by the present invention provides a theoretical basis and technical guarantee for the monitoring, prediction and prevention of TYMaV in the field.

[0010] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0011] Figure 1 It is a design diagram of primers and crRNA in the coat protein gene of TYMaV in Example 1 of the present invention.

[0012] Figure 2 It is an optimization diagram of the reaction time and temperature conditions of RT-RAA and single-tube RT-RAA-CRISPR / Cas12a in Example 1 of the present invention.

[0013] In the figure, (A) RT-RAA temperature optimization. 1: 35 °C; 2: 37 °C; 3: 39 °C; 4: 41 °C.

[0014] (B) RT-RAA reaction time optimization. 1: 10 min; 2: 20 min; 3: 30 min; 4: 40 min; 5: 50 min.

[0015] (C) Optimization of the detection reaction time of single-tube RT-RAA-CRISPR / Cas12a.

[0016] 1: 10 min, 42 °C for 5 min + 37 °C for 5 min; 2: 20 min, 42 °C for 10 min + 37 °C for 10 min; 3: 30 min, 42 °C for 15 min + 37 °C for 15 min; 4: 40 min, 42 °C for 30 min + 37 °C for 10 min; 5: 50 min, 42 °C for 30 min + 37 °C for 20 min; 6: 60 min, 42 °C for 30 min + 37 °C for 30 min; 7: 70 min, 42 °C for 30 min + 37 °C for 40 min; H: RNA of healthy tobacco plants, 42 °C for 30 min + 37 °C for 40 min.

[0017] Figure 3 It is the specific detection of RT-RAA and single-tube RT-RAA-CRISPR / Cas12a in Example 1 of the present invention.

[0018] In the figure, (A) RT-RAA; (B) single-tube RT-RAA-CRISPR / Cas12a 1: TYMaV; 2: TMV; 3: CMV; 4: PVY; 5: ChiVMV; 6: TVBMV; 7: TZSV; 8: TSWV; H: healthy control.

[0019] Figure 4 It is the sensitivity determination of RT-RAA and single-tube RT-RAA-CRISPR / Cas12a in Example 1 of the present invention.

[0020] In the figure, (A) RT-RAA detection.

[0021] (B) RT-PCR detection.

[0022] 1: 1.1×10 7 copies / μL; 2: 1.1×10 6 copies / μL; 3: 1.1×10 5 copies / μL; 4: 1.1×10 4 copies / μL; 5: 1.1×10 3 copies / μL; 6: 1.1×10 2 copies / μL; 7: 1.1×10 1 copies / μL; 8: ddH2O.

[0023] (C) single-tube RT-RAA-CRISPR / Cas12a detection.

[0024] 1: 6.6 ng / μL; 2: 0.66 ng / μL; 3: 66 pg / μL; 4: 6.6 pg / μL; 5: 0.66 pg / μL; 6: 66 fg / μL; 7: 6.6 fg / μL; 8: Healthy control.

[0025] Figure 5 It is the on-site application of RT-RAA and single-tube RT-RAA-CRISPR / Cas12a in Example 1 of the present invention.

[0026] In the figure, (A) RT-RAA detection; (B) single-tube RT-RAA-CRISPR / Cas12a detection.

[0027] 1-4: Tobacco samples to be tested; 5: Positive control; 6: Healthy tobacco samples; 7: Blank control. Specific implementation mode Example

[0028] 1. Design of TYMaV RT-RAA primers, crRNA and probes By aligning the viral sequence of TYMaV (GenBank: OR453866.1) in the GenBank database, RT-RAA primers were designed from the conserved coat protein region of TYMaV ( Figure 1 ), and the specificity of the primers was determined using GenBank. The forward primer TYMaV-F (30 nt) and the reverse primer TYMaV-R (33 nt) (Table 1) can be combined to amplify a target gene fragment of 219 bp. The lateral flow assay reporter gene (LF reporter gene: Famt6-biotin, i.e., the probe TYMaV-LF) and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0029] The PAM site (TTTN or NAAA) is very important for the recognition of the crRNA probe and the target sequence. Therefore, crRNA generally needs to be designed according to the highly conserved region of the existing viral genome in GenBank. In the present invention, crRNA was designed to recognize a specific site in the coat protein region of the TYMaV genome, and this crRNA is located in the homologous region between the binding sequences of the RT-RAA primer pair ( Figure 1). There is a PAM site (GAAA) in the RT-RAA amplicon of TYMaV, and its reverse complementary sequence is TTTC. The corresponding reverse complementary sequence was reverse transcribed as the DNA template for crRNA. The specificity of crRNA was further detected by GenBank BLAST analysis. The primer, crRNA, and probe sequences are shown in (Table 1), and the crRNA was synthesized by General Biosystems (Anhui) Co., Ltd.

[0030] The nucleotide sequence of the tomato yellow mottle-associated virus TYMaV genome is shown in SEQ ID NO:4.

[0031] Table 1 Specific RAA primers, crRNA, and LF probe for detecting TYMaV ; The nucleotide sequence of TYMaV-crRNA in this example is shown in SEQ ID NO:3, which is an RNA sequence. When inputting into the WIPO Squence software, the nucleotide sequence must only contain the symbols listed in Part 1 of Annex I of WIPO ST.26. When the symbol "t" is used without further explanation, it will be interpreted as thymine in DNA and uracil in RNA. The "t" in the SEQ ID NO:3 sequence is actually uracil "U".

[0032] The primer set for detecting the tomato yellow mottle-associated virus TYMaV (tomatoyellow mottle-associated virus, TYMaV) based on RT-RAA-CRISPR / Cas12a in this example includes an RT-RAA amplification primer pair and a CRISPR / Cas12a detection probe; The RT-RAA amplification primer pair includes primer TYMaV-F and primer TYMaV-R, and the nucleotide sequences are shown in SEQ IDNO:1-2; The CRISPR / Cas12a detection probe is TYMaV-crRNA, and the nucleotide sequence is shown in SEQ ID NO:3.

[0033] 2. RNA extraction and crude extraction of virus particles 2.1 Total plant RNA extraction (1) Weigh an appropriate amount of tobacco leaves infected with TYMaV, cut them into pieces and put them into a 2.0 mL centrifuge tube containing steel beads. After quick freezing in liquid nitrogen, grind and shake until it becomes powder; (2) Add 1 mL of Trizol, shake and mix well, and let it stand at room temperature for 5 min; (3) Add 200 μL of chloroform, shake and mix well for 15 s, and let it stand at room temperature for 3 min; (4) Centrifuge at 4 °C and 13,000 g for 15 min. Take a new 1.5 mL centrifuge tube, aspirate 550 μL of the supernatant, add 550 μL of isopropanol, mix well, and let stand at room temperature for 15 min. Then centrifuge at 4 °C and 12,000 g for 10 min, and discard the supernatant. (5) After washing with 1 mL of 75% ethanol, centrifuge at 4 °C and 7,000 g for 5 min, and discard the supernatant. (6) Use a pipette to aspirate all the remaining ethanol and air-dry in a sterile laminar flow hood for 5 - 10 min.

[0034] (7) Add 30 μL of DEPC water to dissolve the precipitate. Measure the total RNA concentration to be 664 - 1170 ng / μL, the A 260 / A 280 ratio is 1.90 - 2.05, and the A 260 / A 230 ratio is 0.83 - 1.06. Store the sample in a -80 °C refrigerator for later use.

[0035] 2.2 Crude extraction of virus particles with PBS buffer Take an appropriate amount of tobacco leaves infected with TYMaV and place them in a self-sealing bag. Add 400 μL of 0.01 M PBS buffer [0.01 M KH2PO4 : 0.01 M Na2HPO4 = 49 : 51 (v / v)]. Rub the sample and let stand for 3 min, then aspirate the supernatant as the crudely extracted virus particles.

[0036] 3. Reverse transcription and construction of plasmid DNA Take 2 μL of the total RNA from TYMaV-infected tobacco (about 2 μg of RNA) or 2 μL of the crudely extracted virus particles as the template, add 4 μL of 4×gDNA wiper Mix and 10 μL of RNase-free ddH2O, mix well, incubate in a water bath at 42 °C for 2 min, add 4 μL of 5×HiScript Ⅲ qRT SuperMix, mix well, incubate in a water bath at 37 °C for 15 min, and then incubate in a water bath at 85 °C for 5 s. Store the obtained cDNA at -20 °C.

[0037] Take the cDNA of TYMaV-infected tobacco, perform PCR amplification using TYMaV-F / TYMaV-R primers. After recovering the PCR product, ligate it to the T vector for sequencing. After correct sequencing, extract plasmid DNA.

[0038] The nucleotide sequence of the primer TYMaV-F is shown in SEQ ID NO:1; The nucleotide sequence of the primer TYMaV-R is shown in SEQ ID NO:2; The reaction system for PCR amplification is as follows: 2xHieff ® 12.5 μL of 2xHieff PCR Master Mix, 0.5 μL of TYMaV-F (10 uM), 0.5 μL of TYMaV-R (10 μM), 1 μL of cDNA, 10.5 μL of ddH2O; The reaction program for PCR amplification is as follows: Pre-denaturation at 95 °C for 3 min; Denaturation at 95 °C for 30 s, Annealing at 55 °C for 15 s, Extension at 72 °C for 30 s, Number of cycles: 32; Final extension at 72 °C for 10 min; The reaction system for ligation is as follows: pEASY ® 1 μL of -T1 Simple Cloning Vector 4 μL of PCR recovery product; The reaction program for ligation is: React at 25 °C for 10 - 15 min.

[0039] 4. Optimization of the RAA detection method The experiment was carried out according to the instructions of the RT-RAA nucleic acid amplification kit (product number: B00R00, Jiangsu Qitian Gene Biotechnology Co., Ltd.). Take a sterile PCR tube to prepare the RAA reaction mixture. The main mixture contains 25 μL of RAA buffer, 16 μL of ddH2O, 2.4 μL of primer TYMaV-F, 2 μL of primer TYMaV-R. After mixing, add it to the RAA reaction tube, gently flick to fully dissolve the recombinant enzyme freeze-dried powder, add 5 μL of 280 mM magnesium acetate solution, and then add 1 μL of plasmid DNA and mix well. Place the PCR tube in a metal bath for reaction. Add an equal volume of phenol-chloroform extraction solution (Tris-saturated phenol: chloroform: isopropanol, volume ratio is 25:24:1), mix well and centrifuge, then take the supernatant for gel electrophoresis detection. The results are as Figure 2 shown in A. When the temperature is 35 °C, 37 °C, 39 °C, 41 °C, target bands with a size of 219 bp appear, and there is no significant difference in the band density. In subsequent experiments, the present invention selects 37 °C as the optimal temperature reaction condition for the RAA reaction. Under the condition of 37 °C, the optimization of the optimal reaction time is carried out. By analyzing the density of the DNA bands, it is found that the DNA yield of the 40-min reaction is almost twice that of the 30-min reaction, and there is no significant difference from that of the 50-min reaction (Figure 2 B). Therefore, the present invention selects a reaction at 37 °C for 40 min as the optimal reaction condition for all conventional RAA detections.

[0040] 5. Optimization of single-tube RT-RAA-CRISPR / Cas12a detection The main mixture is prepared as follows: Add the pre-packaged recombinant enzyme freeze-dried powder in the kit to the RT-RAA reaction tube, and then add 25 μL of RAA buffer, 16 μL of ddH2O, 2.4 μL of primer TYMaV-F, 2.4 μL of primer TYMaV-R, 3 μL of 280 mM magnesium acetate solution, and 4 μL of probe TYMaV-LF to dissolve the enzyme and obtain the RT-RAA reaction mixture; Dispense the evenly mixed RT-RAA reaction mixture into new PCR tubes, with 12.5 μL added to each tube. At the same time, add the Cas12a-mediated CRISPR premix to the PCR tube cap. The CRISPR premix is: 3 μL of NEBuffer3.1 (10×), 1 μL of LbCas12a, 2 μL of TYMaV-crRNA, 1 μL of DTT (dithiothreitol), and 1 μL of RNA inhibitor; Subsequently, add 1 μL of the total RNA or crude virus particles to be tested to the bottom of the PCR tube, gently mix with a pipette, incubate on a 42 °C metal bath for RT-RAA. The RT-RAA reaction product is obtained at the bottom of the PCR tube. After the reaction, mix the CRISPR premix with the RT-RAA reaction product by centrifugation or shaking, and incubate again in a 37 °C metal bath. After the reaction, add 80 μL of ddH2O to each PCR tube, insert the flow-through chromatography test strip into the reaction solution for 5 minutes, and then observe the results. The results show that, as Figure 2 shown in C, when the total time is 20 min (42 °C for 10 min + 37 °C for 10 min), the detection line target band begins to appear. When the total time is above 50 min (42 °C for 30 min + 37 °C for 20 min), the detection line band appears clearly. Therefore, the present invention selects a total reaction time of 50 min (42 °C for 30 min + 37 °C for 20 min) for flow-through chromatography test strip detection based on RT-RAA-CRISPR / Cas12a.

[0041] 6. Specificity analysis of RT-RAA and single-tube RT-RAA-CRISPR / Cas12a detection To verify the specificity of RT-RAA and single-tube RT-RAA-CRISPR / Cas12a flow-through chromatographic test strips, tobacco common RNA viruses such as tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), potato virus Y (PVY), chilli veinal mottle virus (ChiVMV), tobacco veinbanding mosaic virus (TVBMV), tomato zonate spot virus (TZSV), and tomato spotted wilt virus (TSWV) were first detected by RT-PCR for the collected samples to obtain positive samples. Subsequently, the specificity of RT-RAA and single-tube RT-RAA-CRISPR / Cas12a flow-through chromatographic test strips for detecting TYMaV was verified. After performing the RAA reaction using the cDNA of tobacco plants infected with TYMaV, TMV, CMV, PVY, ChiVMV, TVBMV, TZSV, TSWV, and healthy tobacco plants as templates, the RAA products were purified and detected by gel electrophoresis. The results showed that only the tobacco samples infected with TYMaV could amplify the target band, while other samples did not amplify the target band. At the same time, in the detection of single-tube RT-RAA-CRISPR / Cas12a flow-through chromatographic test strips, only the samples infected with TYMaV showed positive bands, which was consistent with the results of RAA detection ( Figure 3 ). These results indicate that the TYMaV detection system designed in the present invention has strong specificity.

[0042] 7. Sensitivity analysis of the TYMaV detection method The plasmid of the TYMaV fragment with a concentration of 56.7 ng / μL was diluted in concentration and the copy number was calculated. The converted concentrations were 1.1×10 7 copies / μL, 1.1×10 6 copies / μL, 1.1×10 5 copies / μL, 1.1×10 4 copies / μL, 1.1×10 3 copies / μL, 1.1×10 2 copies / μL, 1.1×10 1copies / μL. The above samples were subjected to RAA and PCR detection. The results showed that the RAA method could detect the TYMaV fragment plasmid at a concentration of 1.1×10 2 copies / μL, which was 10 times more sensitive than the PCR method ( Figure 4 A, B).

[0043] Total RNA was extracted from tobacco leaves infected with TYMaV, and the concentration was 664 ng / μL. Its concentration was serially diluted to 6.6 ng / μL, 0.66 ng / μL, 66 pg / μL, 6.6 pg / μL, 0.66 pg / μL, 66 fg / μL, 6.6 fg / μL. Different concentration samples were detected by a single-tube RT-RAA-CRISPR / Cas12a flow-through chromatographic test strip. The results showed that the single-tube RT-RAA-CRISPR / Cas12a flow-through chromatographic test strip could detect TYMaV in samples with an RNA concentration of 66 fg / μL ( Figure 4 C), with high sensitivity.

[0044] 8. Field application of the TYMaV detection method To verify the feasibility of the developed single-tube RT-RAA-CRISPR / Cas12a lateral flow chromatographic test strip technology, tobacco samples suspected of virus infection were randomly collected in southwestern Guizhou. After grinding the tobacco leaves, 400 μL of 0.01 M PBS buffer was added, and crude virus particles were used for RT-RAA (reaction conditions: 37 °C for 40 min) and single-tube RT-RAA-CRISPR / Cas12a (RT-RAA reaction at 42 °C for 30 min, CRISPR / Cas12a reaction at 37 °C for 20 min) lateral flow chromatographic test strip detection. The RT-RAA detection results showed that the target band could be amplified in sample 3, proving the feasibility of using crude virus particles by RT-RAA to detect TYMaV ( Figure 5 A). The results of the single-tube RT-RAA-CRISPR / Cas12a lateral flow chromatography test showed that a positive band was detected in sample 3, which was consistent with the RT-RAA results, further verifying the feasibility of the single-tube RT-RAA-CRISPR / Cas12a lateral flow chromatographic test strip method for detecting TYMaV in the field ( Figure 5 B).

[0045] This example provides a method for visual and rapid detection of tomato yellow mottle-associated virus TYMaV, and the method is as follows: S1. Extract the total RNA of tobacco leaves infected with tomato yellow mottle-associated virus TYMaV to obtain total RNA; Alternatively, the viral particles of tobacco leaves infected with tomato yellow mottle-associated virus TYMaV were crudely extracted with PBS buffer to obtain crudely extracted viral particles; S2. Prepare the RT-RAA reaction mixture: Add the pre-packaged recombinant enzyme lyophilized powder in the kit to the RT-RAA reaction tube, and then add 25 μL of RAA buffer, 16 μL of ddH2O, 2.4 μL of primer TYMaV-F, 2.4 μL of primer TYMaV-R, 3 μL of 280 mM magnesium acetate solution, and 4 μL of probe TYMaV-LF to dissolve the enzyme and obtain the RT-RAA reaction mixture; The sequence of the probe TYMaV-LF is Biotin-TTTTTT-FAM; S3. Prepare the CRISPR premix: The system of the CRISPR premix is: 3 μL of 10×NEBuffer3.1, 1 μL of LbCas12a, 2 μL of TYMaV-crRNA, 1 μL of dithiothreitol, and 1 μL of RNA inhibitor; S4. Add the RT-RAA reaction mixture obtained in S2 to the bottom of the PCR tube, then add 1 μL of the total RNA or crudely extracted viral particles obtained in S1, and then place the CRISPR premix obtained in S3 in the lid of the PCR tube. Incubate in a metal bath at 42 °C for 30 min, then place the CRISPR premix in the lid of the tube at the bottom of the tube by centrifugation or shaking to mix with the reaction product at the bottom of the tube. Incubate in a metal bath at 37 °C for 20 min, then add 80 μL of ddH2O, and detect the target band with a flow-through chromatography test strip; if both the test line and the control line show red bands, the sample is positive; if only the control line shows a red band, the sample is negative; if the control line does not show a band, the test strip is invalid.

[0046] The primer set and the visual rapid detection method for detecting tomato yellow mottle-associated virus based on RT-RAA-CRISPR / Cas12a in this example can be used to prepare a kit for detecting tomato yellow mottle-associated virus TYMaV.

[0047] The above is only a preferred embodiment of the present invention and does not impose any limitations on the present invention. Any simple modifications, changes, and equivalent variations made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A primer set for detecting Tomato yellow mottle-associated virus TYMaV based on RT-RAA-CRISPR / Cas12a, characterized in that, It includes an RT-RAA amplification primer pair and a CRISPR / Cas12a detection probe; The RT-RAA amplification primer pair includes primer TYMaV-F and primer TYMaV-R; The nucleotide sequence of the primer TYMaV-F is shown as SEQ ID NO:1; The nucleotide sequence of the primer TYMaV-R is shown as SEQ ID NO:2; The CRISPR / Cas12a detection probe is TYMaV-crRNA, and the nucleotide sequence is shown as SEQ ID NO:

3.

2. A method for visual and rapid detection of tomato yellow mottle-associated virus TYMaV using the primer set for detecting tomato yellow mottle-associated virus TYMaV based on RT-RAA-CRISPR / Cas12a as described in claim 1, characterized in that, The method is as follows: S1. Extract the total RNA of tobacco leaves infected with Tomato yellow mottle-associated virus TYMaV to obtain the total RNA; Or roughly extract the virus particles of tobacco leaves infected with Tomato yellow mottle-associated virus TYMaV with PBS buffer to obtain the roughly extracted virus particles; S2. Prepare the RT-RAA reaction mixture: Add the recombinant enzyme freeze-dried powder to the RT-RAA reaction tube, and then add 25 μL of RAA buffer, 16 μL of ddH2O, 2.4 μL of primer TYMaV-F, 2.4 μL of primer TYMaV-R, 3 μL of 280 mM magnesium acetate solution, and 4 μL of probe TYMaV-LF, and dissolve the enzyme to obtain the RT-RAA reaction mixture; The sequence of the probe TYMaV-LF is Biotin-TTTTTT-FAM; S3. Prepare the CRISPR premix: The system of the CRISPR premix is: 3 μL of 10×NEBuffer3.1, 1 μL of LbCas12a, 2 μL of TYMaV-crRNA, 1 μL of dithiothreitol, 1 μL of RNA inhibitor; S4. Add the RT-RAA reaction mixture obtained in S2 to the bottom of the PCR tube, then add 1 μL of the total RNA or the roughly extracted virus particles obtained in S1, and then place the CRISPR premix obtained in S3 inside the PCR tube cap. Incubate in a metal bath at 42 °C for 30 min, and the RT-RAA reaction product is obtained at the bottom of the tube; Place the CRISPR premix inside the tube cap at the bottom of the tube by centrifugation or shaking, mix it with the RT-RAA reaction product at the bottom of the tube, incubate in a metal bath at 37 °C for 20 min, then add 80 μL of ddH2O, and detect the target band with a flow-through chromatography test strip.

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