RT-LAMP primer group for detecting cherry virus A and application of RT-LAMP primer group

Abstract

The invention belongs to the technical field of molecular biological detection, and particularly relates to an RT-LAMP primer group for detecting cherry virus A and application of the RT-LAMP primer group. According to the RT-LAMP primer group disclosed by the invention, the rapid and high-specificity amplification of the genome of the cherry virus A (CVA) can be realized. The RT-LAMP detection method established on the basis of the RT-LAMP primer group shows extremely high sensitivity, and the detection sensitivity to CVA cDNA is remarkably improved compared with that of a conventional RT-PCR method. Meanwhile, the RT-LAMP primer group disclosed by the invention has excellent specificity, and has no cross reaction with five common cherry viruses, so that the accuracy of a detection result is ensured. More importantly, the whole detection process of the RT-LAMP detection method established on the basis of the RT-LAMP primer group can be completed within 40 min, and the RT-LAMP detection method has the advantages of being easy and convenient to operate and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to an RT-LAMP primer set for detecting cherry virus A and its application. Background Technology

[0002] Cherry Virus A ( Cherry Virus A CVA is one of the main viruses that infect cherry and other plum species. It has a wide host range and is widely distributed. It is the dominant virus that infects both sweet and sour cherries.

[0003] Currently, various methods such as ELISA and RT-PCR are used for the detection of CVA. However, the ELISA method is easily affected by antibody quality and fruit tree tissue composition, which may lead to false negative results; while the RT-PCR method strongly relies on a high concentration of initial template, making it difficult to achieve effective detection in samples with low viral load, and thus failing to meet the timeliness requirements for rapid field monitoring and early warning. Summary of the Invention

[0004] The purpose of this invention is to solve the aforementioned problems existing in the prior art and to provide a CVA detection technology with high specificity and sensitivity. To this end, this invention provides an RT-LAMP primer set for detecting cherry virus A and its application.

[0005] This invention provides an RT-LAMP primer set for detecting cherry virus A, the RT-LAMP primer set comprising outer primer F3 as shown in SEQ ID NO.1, outer primer B3 as shown in SEQ ID NO.2, inner primer FIP as shown in SEQ ID NO.3, and inner primer BIP as shown in SEQ ID NO.4.

[0006] Preferably, the RT-LAMP primer set further includes the circular primer LB as shown in SEQ ID NO.5.

[0007] The present invention also provides a reagent or kit for detecting cherry virus A, comprising the RT-LAMP primer set described in the above technical solution.

[0008] This invention also provides the application of the RT-LAMP primer set or the reagent or kit described in the above technical solution in the cultivation of virus-free seedlings and / or seedling quarantine.

[0009] Preferably, the seedling quarantine includes the detection of cherry virus A.

[0010] This invention also provides a method for detecting cherry virus A using RT-LAMP, comprising the following steps: 1) Extract total RNA from the sample to be tested; reverse transcribe the total RNA to obtain cDNA; 2) Using the cDNA as a template, perform RT-LAMP amplification using the RT-LAMP primer set described in the above technical solution. If a specific amplification product is detected, determine that the sample contains cherry virus A; or, include the following steps: (1) Extract total RNA from the sample to be tested; (2) Using the total RNA as a template, reverse transcription and RT-LAMP amplification of RNA are performed sequentially in the same reaction system. The RT-LAMP amplification is performed using the RT-LAMP primer set described in the above technical scheme. If a specific amplification product is detected, it is determined that the sample to be tested contains cherry virus A.

[0011] Preferably, the reaction conditions for RT-LAMP amplification in step 2) are 60-70℃ incubation for 30-50 min; The RT-LAMP amplification reaction system in step 2) is 25 μL and includes the following components: 2.5 μL of 10×Thermopol reaction buffer, 4.0 μL of 10 μmol / L inner primer, 1.0 μL of 10 μmol / L outer primer, 0.4 μL of 10 μmol / L loop primer, 1.5 μL of 100 mM MgSO4 solution, 1 μL of 5M betaine, 2.5 μL of 10 mM dNTPs Mix, 1.0 μL of 8000 U / mL Bst 2.0 DNA polymerase, 1.5 μL of template, and ddH2O to a final volume of 25 μL.

[0012] Preferably, the reaction system in step (2), in 25 μL, comprises the following components: 1 μL of 200 U / μL M-MLV Reverse Transcriptase, 4 μL of 5×M-MLV reaction buffer, 2.5 μL of 10×Thermopol reaction buffer, 4.0 μL of 10 μmol / L inner primer, 1.0 μL of 10 μmol / L outer primer, 1 μL of 10 μmol / L loop primer, 1.5 μL of 100 mM MgSO4 solution, 1 μL of 5 M betaine, 2.5 μL of 10 mM dNTPs Mix, 1.0 μL of 8000 U / mL Bst 2.0 DNA polymerase, 1.5 μL of template, and ddH2O to bring the total to 25 μL.

[0013] Preferably, the reaction conditions for reverse transcription of RNA in step (2) are: first, treatment at 42°C for 15 min, followed by treatment at 90°C for 3 min; The reaction conditions for RT-LAMP amplification in step (2) are 60~70℃ for 30~50 min.

[0014] Preferably, the detection method for the specific amplification product includes electrophoresis detection and / or fluorescent dye colorimetric detection; If the electrophoresis results show waterfall-like trapezoidal characteristic bands, it is determined that the sample contains cherry virus A. If the result of the fluorescent dye colorimetric detection is fluorescence, then it is determined that the sample contains cherry virus A. The fluorescent dye includes SYBR Green I.

[0015] Beneficial effects: This invention provides an RT-LAMP primer set for detecting cherry virus A. The RT-LAMP primer set of this invention includes outer primer F3 as shown in SEQ ID NO.1, outer primer B3 as shown in SEQ ID NO.2, inner primer FIP as shown in SEQ ID NO.3, and inner primer BIP as shown in SEQ ID NO.4. The RT-LAMP primer set of this invention enables rapid and highly specific amplification of the cherry virus A (CVA) genome based on the principle of loop-mediated isothermal amplification. The RT-LAMP detection method established based on the RT-LAMP primer set of this invention exhibits extremely high sensitivity, with a detection limit for CVA cDNA as low as 675.4 × 10⁻⁶. -4 ng / μL, with sensitivity higher than conventional RT-PCR methods (detection limit of 10). -2 The detection rate (ng / μL) was significantly improved. Simultaneously, the RT-LAMP primer set described in this invention exhibits excellent specificity, showing no cross-reactivity with any of the five common cherry viruses, ensuring the accuracy of the detection results. More importantly, the entire detection process of the RT-LAMP detection method based on the RT-LAMP primer set described in this invention can be completed within 40 minutes, and the detection results can be directly interpreted visually after staining with SYBR Green I fluorescent dye, offering advantages such as ease of operation and low cost. Validated on 70 field samples, the RT-LAMP detection method based on the RT-LAMP primer set described in this invention achieved a detection rate of 91.42% for cherry virus A, demonstrating strong field applicability and providing an efficient and reliable technical tool for screening sweet cherry seedlings for virus infection, early warning of CVA, and prevention and control of virus transmission. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1Figure showing the alignment analysis results of the CVA genome sequence using BioEdit software; Figure 2 This is a graph showing the RT-LAMP specificity detection results for CVA; where, Figure 2 In the image, A represents the result of gel electrophoresis. Figure 2 M in the text represents the 2000 DNA marker lane. Figure 2 The 1 in the text represents the amplification product lane of the CVA sample. Figure 2 Lane 2 in the text represents the amplification product lanes of the PDV sample. Figure 2 Lane 3 in the image represents the amplification product lanes of the PNRSV sample. Figure 2 Lane 4 in the image represents the amplification product lanes of the CGRMV sample. Figure 2 Lane 5 in the image represents the amplification products of the CRLV sample. Figure 2 Lane 6 in the image represents the amplification products of the LChV-1 sample. Figure 2 In this context, NC represents the amplification product lane of the virus-free tissue culture control. Figure 2 In the image, 'a' represents the fluorescence visualization result, which corresponds to the sample represented by the lane above. Figure 3 The graph shows the CVA detection results for RT-LAMP reaction systems with different dNTP contents; among them, Figure 3 In the image, A represents the result of gel electrophoresis. Figure 3 M in the text represents the 2000 DNA marker lane. Figure 3 In the figures 1-6, the amplification product lanes are shown for RT-LAMP reaction systems with dNTP concentrations of 0 mmol / L, 0.5 mmol / L, 1.0 mmol / L, 1.5 mmol / L, 2.0 mmol / L, and 2.5 mmol / L, respectively. Figure 3 NC in the sample is a negative control. Figure 3 B in the image represents the fluorescence visualization result, which corresponds to the sample represented by the lane above. Figure 4 The graph shows the CVA detection results for RT-LAMP reaction systems with different betaine contents; among them, Figure 4 In the image, A represents the result of gel electrophoresis. Figure 4 M in the text represents the 2000 DNA marker lane. Figure 4 In the diagram, 1-6 represent the amplification product lanes in the RT-LAMP reaction system when the betaine concentration is 0 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, and 1.0 mol / L, respectively. Figure 4 NC in the sample is a negative control. Figure 4 B in the image represents the fluorescence visualization result, which corresponds to the sample represented by the lane above. Figure 5 For different Mg 2+ The CVA detection results of the RT-LAMP reaction system are shown in the figure; among them, Figure 5 In the image, A represents the result of gel electrophoresis. Figure 5 M in the text represents the 2000 DNA marker lane. Figure 5 1 to 6 represent the Mg in the RT-LAMP reaction system. 2+ The amplification product lanes at concentrations of 0 mmol / L, 2 mmol / L, 4 mmol / L, 6 mmol / L, 8 mmol / L, and 10 mmol / L. Figure 5 NC in the sample is a negative control. Figure 5 B in the image represents the fluorescence visualization result, which corresponds to the sample represented by the lane above. Figure 6 The graph shows the CVA detection results for RT-LAMP reaction systems with different Bst2.0 DNA polymerase contents; among them, Figure 6 In the image, A represents the result of gel electrophoresis. Figure 6 M in the text represents the 2000 DNA marker lane. Figure 6 In the diagram, 1-6 represent the amplification product lanes in the RT-LAMP reaction system when the Bst2.0 DNA polymerase concentration is 0.128 U / μL, 0.192 U / μL, 0.256 U / μL, 0.320 U / μL, 0.384 U / μL, and 0.448 U / μL, respectively. Figure 6 NC in the sample is a negative control. Figure 6 B in the image represents the fluorescence visualization result, which corresponds to the sample represented by the lane above. Figure 7 The graph shows the CVA detection results for RT-LAMP reaction systems with different ratios of inner and outer primers and different concentrations of loop primers; among them, Figure 7 Figure A in the graph shows the gel electrophoresis results of RT-LAMP reaction systems with different ratios of inner and outer primers. Figure 7 M in the text represents the 2000 DNA marker lane. Figure 7 In Figure A, lanes 1-6 represent the amplification products in the RT-LAMP reaction system when the ratios of inner and outer primers are 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1, respectively. Figure 7 Figure A shows 8 negative controls. Figure 7 B in the figure represents the fluorescence visualization results of RT-LAMP reaction systems with different ratios of inner and outer primers, which correspond to the samples represented by the lanes above. Figure 7 In the figure, C represents the gel electrophoresis detection results of RT-LAMP reaction systems with different ring primer concentrations. Figure 7M in the text represents the 2000 DNA marker lane. Figure 7 In Figure C, lanes 1-6 represent the amplification products in the RT-LAMP reaction system at concentrations of 0.0 μmol / L, 0.4 μmol / L, 0.6 μmol / L, 0.8 μmol / L, 1.0 μmol / L, and 1.2 μmol / L, respectively. Figure 7 The 8 in Figure C are negative controls. Figure 7 D in the figure represents the fluorescence visualization results of RT-LAMP reaction systems with different ring primer concentrations, which correspond to the samples represented by the lanes above. Figure 8 The graph shows the CVA detection results for RT-LAMP reaction systems at different reaction temperatures and times; among them, Figure 8 In the figure, A represents the gel electrophoresis detection results of the RT-LAMP reaction system at different reaction temperatures. Figure 8 M in the text represents the 2000 DNA marker lane. Figure 8 In Figure A, lanes 1-8 represent the amplification products of the RT-LAMP reaction system at reaction temperatures of 58℃, 59℃, 60℃, 62℃, 65℃, 67℃, 69℃, and 70℃, respectively. Figure 8 B in the figure represents the fluorescence visualization results of the RT-LAMP reaction system at different reaction temperatures, which correspond to the samples represented by the lanes above. Figure 8 In the figure, C represents the gel electrophoresis detection results of the RT-LAMP reaction system at different reaction times. Figure 8 M in the text represents the 2000 DNA marker lane. Figure 8 In Figure C, lanes 1-6 represent the amplification products at reaction times of 25 min, 30 min, 35 min, 40 min, 45 min, and 50 min, respectively. Figure 8 The NC in the middle C figure is the negative control. Figure 8 D in the figure represents the fluorescence visualization results of RT-LAMP reaction systems with different ring primer concentrations, which correspond to the samples represented by the lanes above. Figure 9 The graph shows the CVA detection results for different template dilution factors; among them, Figure 9 In the image, A represents RT-PCR gel electrophoresis images at different template dilutions. Figure 9 In Figure A, lane M represents the 2000 DNA marker lane. Figure 9 Figure A shows that numbers 1-7 represent the template cDNA content of 675.4 × 10⁻⁶. 0 ng / µL ~675.4×10 -6 The amplification product lanes of RT-PCR at ng / µL Figure 9NC in Figure A represents the negative control; Figure 9 In the image, B represents RT-LAMP gel electrophoresis images with different template dilution factors. Figure 9 In Figure B, lane M represents the 2000 DNA marker lane. Figure 9 Figure B shows that numbers 1-7 represent the template cDNA content of 675.4 × 10⁻⁶. 0 ng / µL ~675.4×10 -6 The amplification product lanes of RT-LAMP at ng / µL Figure 9 The NC in Figure B represents the negative control; Figure 9 In the figure, b represents the fluorescence visualization results of RT-LAMP reaction systems with different template dilution factors, which correspond to the samples represented by the lanes above. Figure 10 The figure shows the specificity verification results of the RT-LAMP system; where, Figure 10 In the image, A represents an RT-LAMP gel electrophoresis image. Figure 10 In Figure A, lane M represents the 2000 DNA marker lane. Figure 10 Lanes 1-3 in Figure A represent the amplification products of CVA. Figure 10 Lanes 4-6 in Figure A are the amplification products of PDV; Figure 10 Lanes 7-9 in Figure A are the amplification products of PNRSV; Figure 10 Lanes 10-12 in Figure A represent the amplification products of CGRMV; Figure 10 Lanes 13-15 in Figure A are the amplification products of CRLV; Figure 10 Lanes 16-18 in Figure A are the amplification products of LChV-1; Figure 10 Lane 19 in Figure A represents the amplification product of the virus-free tissue culture seedlings; Figure 10 In Figure A, lanes 20 and 21 represent the amplification products of DEPC-ddH2O. Figure 9 The RT-LAMP fluorescence visualization results are shown in the figure, and correspond to the samples represented by the lanes above. Figure 11 The figure shows the results of the nucleic acid crude extraction process and the validation of the RT-LAMP system; among them, Figure 11 (1) to (4) are flowcharts of the crude nucleic acid extraction process; Figure 11 Figure (5) shows the results of the RT-LAMP analysis. Figure 10 In section (5), A represents the cDNA template color development result. Figure 10 B in (5) is the color development result of the crude nucleic acid extract template; Figure 12 Visualization of RT-PCR and RT-LAMP results from 70 sweet cherry samples; among them, Figure 12In this context, A represents the RT-PCR test result. Figure 12 In Figure A, lane M represents the 2000 DNA marker lane. Figure 12 In Figure A, lanes 1-70 represent the amplification products of 70 sweet cherry samples. Figure 12 71 in Figure A represents the amplification lane of the virus-free tissue culture seedling sample. Figure 12 72 in Figure A is the negative control (ddH2O); Figure 12 B in the image represents the RT-LAMP fluorescence visualization detection result, which corresponds to the sample represented by the lane above. Figure 13 The images show the fluorescence colorimetric results after LAMP reactions using crude nucleic acid extracts of different concentrations as templates; among them... Figure 13 Figures 1-6 show the fluorescence colorimetric results at dilution ratios of 1-6. Figure 13 The NC figure shows the fluorescence color development results of virus-free tissue culture seedlings. Detailed Implementation

[0018] This invention provides an RT-LAMP primer set for detecting cherry virus A, comprising outer primer F3 as shown in SEQ ID NO.1, outer primer B3 as shown in SEQ ID NO.2, inner primer FIP as shown in SEQ ID NO.3, and inner primer BIP as shown in SEQ ID NO.4. As one embodiment, the RT-LAMP primer set of this invention further includes a loop primer LB as shown in SEQ ID NO.5. As one embodiment, the RT-LAMP primer set of this invention can achieve rapid and highly specific amplification of the cherry virus A (CVA) genome based on the principle of loop-mediated isothermal amplification.

[0019] This invention also provides a reagent or kit for detecting cherry virus A, comprising the RT-LAMP primer set described in the above-described technical solution. As one embodiment, the kit of this invention further comprises other RT-LAMP amplification-related reagents and reagents for detecting amplification products. As one embodiment, the reagents for detecting amplification products of this invention include electrophoresis detection reagents and / or fluorescent dye colorimetric detection reagents, wherein the fluorescent dye includes SYBR Green I.

[0020] This invention also provides the application of the RT-LAMP primer set or reagents or kits described in the above-mentioned technical solutions in virus-free seedling cultivation and / or seedling quarantine. As one embodiment, the seedling quarantine described in this invention includes the detection of cherry virus A. As one embodiment, the RT-LAMP primer set, reagents, or kits described in this invention exhibit strong field applicability, providing an efficient and reliable technical tool for screening sweet cherry seedlings for virus infection, early warning of CVA, and prevention and control of virus transmission.

[0021] This invention also provides a method for detecting cherry virus A using RT-LAMP, comprising the following steps: 1) Extract total RNA from the sample to be tested; reverse transcribe the total RNA to obtain cDNA; 2) Using the cDNA as a template, perform RT-LAMP amplification using the RT-LAMP primer set described in the above technical solution. If a specific amplification product is detected, determine that the sample contains cherry virus A; or, include the following steps: (1) Extract total RNA from the sample to be tested; (2) Using the total RNA as a template, reverse transcription and RT-LAMP amplification of RNA are performed sequentially in the same reaction system. The RT-LAMP amplification is performed using the RT-LAMP primer set described in the above technical scheme. If a specific amplification product is detected, it is determined that the sample to be tested contains cherry virus A.

[0022] When performing RT-LAMP detection of cherry virus A according to steps 1) and 2): This invention extracts total RNA from the sample to be tested; the total RNA is then reverse transcribed to obtain cDNA. As one embodiment, the reverse transcription system of this invention is 5... FastKing-RTSuperMix 4 μL, Total RNA 1 μL (50 ng ~ 2 μg), ddH2O to make up to 20 μL. As one embodiment, the reverse transcription reaction procedure of this invention is: first react at 42℃ for 15 min, then react at 95℃ for 3 min.

[0023] After obtaining the cDNA, the present invention uses the cDNA as a template and performs RT-LAMP amplification using the RT-LAMP primer set described in the above technical solution. If a specific amplification product is detected, it is determined that the sample contains cherry virus A. As one embodiment, the dNTP content in the RT-LAMP amplification reaction system of the present invention can be 1.0~2.5 mmol·L⁻¹. -1As one embodiment, the betaine content in the RT-LAMP amplification reaction system of the present invention can be 0~0.4 mol / L. The MgSO4 solution in the RT-LAMP amplification reaction system of the present invention contains Mg... 2+ The concentration can be 2-10 mmol / L. The Bst2.0 DNA polymerase concentration in the RT-LAMP amplification reaction system of the present invention can be 8.0-11.2 U / μL. The circular primer concentration in the RT-LAMP amplification reaction system of the present invention can be 0.0 μmol / L-1.2 μmol / L. As one embodiment, the final concentration ratio of inner primer FIP and inner primer BIP to the final concentration ratio of outer primer F3 and outer primer B3 in the RT-LAMP primer set of the present invention is (1-10):1. In one embodiment, the RT-LAMP amplification reaction system in step 2) of the present invention, in 25 μL volume, comprises the following components: 2.5 μL of 10×Thermopol reaction buffer, 4.0 μL of 10 μmol / L inner primer, 1.0 μL of 10 μmol / L outer primer, 1 μL of 10 μmol / L loop primer, 1.5 μL of 100 mM MgSO4 solution, 1 μL of 5 M betaine, 2.5 μL of 10 mM dNTPs Mix, 1.0 μL of 8000 U / mL Bst 2.0 DNA polymerase, 1.5 μL of template, and ddH2O to a final volume of 25 μL. In another embodiment, the RT-LAMP amplification reaction conditions in step 2) of the present invention are incubation at 60–70 °C for 30–50 min.

[0024] When performing RT-LAMP detection of cherry virus A according to steps (1) and (2): This invention extracts total RNA from the sample to be tested. As one embodiment, the total RNA from the sample to be tested can be obtained using a lysis-dilution method.

[0025] After extracting total RNA from the sample to be tested, this invention uses the total RNA as a template to sequentially perform reverse transcription and RT-LAMP amplification in the same reaction system. The RT-LAMP amplification is performed using the RT-LAMP primer set described in the above technical scheme. If a specific amplification product is detected, it is determined that the sample to be tested contains cherry virus A. As one embodiment, the dNTP content in the RT-LAMP amplification reaction system of this invention can be 1.0~2.5 mmol·L⁻¹. -1 As one embodiment, the betaine content in the RT-LAMP amplification reaction system of the present invention can be 0~0.4 mol / L. The MgSO4 solution in the RT-LAMP amplification reaction system of the present invention contains Mg... 2+The concentration can be 2-10 mmol / L. The Bst2.0 DNA polymerase concentration in the RT-LAMP amplification reaction system of the present invention can be 8.0-11.2 U / μL. The circular primer concentration in the RT-LAMP amplification reaction system of the present invention can be 0.0 μmol / L-1.2 μmol / L. As one embodiment, the final concentration ratio of inner primer FIP and inner primer BIP to the final concentration ratio of outer primer F3 and outer primer B3 in the RT-LAMP primer set of the present invention is (1-10):1. As one embodiment, the reaction system of step (2) of the present invention, in 25 μL, includes the following components: 1 μL of 200 U / μL M-MLV Reverse Transcriptase, 4 μL of 5×M-MLV reaction buffer, 2.5 μL of 10×Thermopol reaction buffer, 4.0 μL of 10 μmol / L inner primer, 1.0 μL of 10 μmol / L outer primer, 1 μL of 10 μmol / L loop primer, 1.5 μL of 100 mM MgSO4 solution, 1 μL of 5M betaine, 2.5 μL of 10 mM dNTPs Mix, 1.0 μL of 8000 U / mL Bst 2.0 DNA polymerase, 1.5 μL of template, and ddH2O to a final volume of 25 μL. In one embodiment, the reverse transcription reaction conditions for RNA in step (2) of the present invention are: first, treatment at 42℃ for 15 min, followed by treatment at 90℃ for 3 min; the RT-LAMP amplification reaction conditions in step (2) are: incubation at 60~70℃ for 30~50 min. In one embodiment, when performing RT-LAMP detection of cherry virus A according to steps (1) and (2), the present invention can perform rapid CVA detection on the crude RNA extracted from the sample to be tested in the same reaction system. In one embodiment, the results of RT-LAMP detection of the crude RNA extracted from the sample to be tested are consistent with the cDNA template detection results. In one embodiment, the present invention obtains the sample template through the crude nucleic acid extraction method, simplifying the molecular detection process of plant viruses and shortening the sample template acquisition time to approximately 15 min, further accelerating the overall process of RT-LAMP detection of CVA.

[0026] As one implementation method, the detection method for the specific amplification product of the present invention includes electrophoresis detection and / or fluorescent dye colorimetric detection; if the electrophoresis detection result shows a waterfall-like trapezoidal characteristic band, it is determined that the sample contains cherry virus A; if the fluorescent dye colorimetric detection result shows fluorescence, it is determined that the sample contains cherry virus A; the fluorescent dye includes SYBR Green I. As one implementation method, the detection sensitivity of the method for detecting cherry virus A of the present invention can reach 675.4 × 10⁻⁶. -4ng·µL -1 Much higher than the 675.4 × 10⁻⁶ of conventional RT-PCR. -2 ng·µL -1 As one implementation method, the method for detecting cherry virus A described in this invention has a detection rate of up to 91.42% for CVA-positive sweet cherry leaf samples, which is much higher than the detection rate of conventional RT-PCR (71.42%).

[0027] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an RT-LAMP primer set for detecting cherry virus A and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0028] The cherry virus A used in the embodiments of the present invention ( Cherry virus A (CVA) Prunus necrotic ringspot virus Prunus necrotic ringspot virus (PNRSV), Li dwarf virus ( Prune dwarf virus PDV), cherry leaf scraper virus (PDV) Cherry raspberry leaf virus CRLV) Cherry Green Ring Mottle Virus ( Cherry green ring mottle virus CGRMV) and cherry fruit virus No. 1 ( Little cherry virus 1. All samples (LChV-1) were prepared as cDNA templates by the laboratory after RT-PCR amplification of the target fragment and verification by agarose gel electrophoresis, and stored at -80℃ for later use.

[0029] The test reagents used in this invention were obtained from the following sources: the Polysaccharide & Polyphenolic Rich RNA Extraction Kit (PoLysaccharide-rides & Polyphenolics-rich RNAprep Pure) and the FastKing one-step genomic cDNA first-strand synthesis premix (FastKing gDNA Dispelling RT SuperMix) were purchased from Tiangen Biotech (Beijing) Co., Ltd. Isothermal amplification 2×RT-LAMP Master Mix premix and dNTPs (10 mM·L⁻¹) were also used. -1 Shanghai Sangon Biotech Co., Ltd. M-MLV reverse transcriptase (10000 U / mL), MgSO4 (100 mmol·L⁻¹) -1 Betaine and Bst2.0 Warm Start® DNA polymerase: New England Biolabs, USA. 1000×SYBR Green I dye: Beijing Solarbio Technology Co., Ltd.

[0030] The main instruments in this embodiment of the invention include a gradient ABV-PCR instrument, a micro-volume nucleic acid analyzer, Bio-Rad, a clean bench, a high-speed benchtop micro-volume refrigerated centrifuge, a DYCZ 300 electrophoresis apparatus, a Tanon 2500 gel imaging system, and an electronic balance.

[0031] Example 1 RT-LAMP primer design and amplification effect verification 1. RT-LAMP primer design Download the CVA genome sequence (accession number: NC003689, OR148948.1, ON216678.1, ON216675.1, ON216674, ON216676.1) and the shell protein of the CVA isolates accessed in GenBank. Coat Protein The conserved regions of the nucleotide sequence of the CP gene (accession number: ON216677.1) were analyzed using BioEdit software. The alignment results are as follows: Figure 1 As shown in Table 1, a relatively conservative fragment with small variation was selected as a template. Three sets of specific primers for RT-LAMP detection were designed online using the RT-LAMP primer design online software PrimerExplorerV5 (http: / / primerexplorer.Jp / RT-LAMPv5e / index.html). RT-PCR detection primers were also designed simultaneously.

[0032] Table 1. Specific primers for RT-LAMP and RT-PCR of cherry virus A.

[0033] 2. Verification of RT-LAMP primer amplification effect (1) RNA extraction and cDNA synthesis Total RNA was extracted from CVA-infected sweet cherry samples using a polysaccharide and polyphenol plant total RNA extraction kit. 2.5 μL of the RNA stock solution was subjected to 1.5% agarose gel electrophoresis. The integrity and brightness ratio of the 28S / 18S bands were detected using a gel imaging system, and the concentration and absorbance (A260 / A280) were measured using a micro-nucleic acid analyzer to assess RNA quality. Using this RNA as a template, cDNA was synthesized by reverse transcription using the FastKing one-step genomic DNA removal kit. (Reverse transcription system: 5...) FastKing-RTSuperMix 4μL, Total RNA 1μL (50ng~2μg), ddH2O to make up to 20μL; Reaction program: react at 42℃ for 15min first, then at 95℃ for 3min. Store the product at -20℃ for later use.

[0034] (2) RT-LAMP primer screening Primers were obtained using the above design, and CVA was amplified using cDNA from CVA-positive plants as a template. The reaction mixture (25 μL) included the following components: 12.5 μL of universal 2×RT-LAMP Master Mix, CVA-FIB / BIP (10 μmol·L⁻¹). -1 ) 4.0 μL, CVA-F3 / B3 (10 μmol·L -1 ) 1 μL, CVA-LB (10 μmol·L -1 1.0 μL of SYBR Green I, 0.5 μL of DNA Polymerase, 0.5 μL of SYBR Green I, 1 μL of Template cDNA (675.4 ng), and ddH2O to make up the volume. Reaction conditions: 65℃ for 45 min. Healthy tissue-cultured cherry seedlings were included as a negative control for each primer group. After the reaction, 2.5 μL of the product was subjected to agarose gel electrophoresis, and the ladder-like bands were observed using a gel imaging system. Additionally, 0.5 μL of SYBR Green I nucleic acid dye was added to the inner cap of the reaction tube, and the mixture was briefly centrifuged and observed for color: fluorescent green indicated a positive result, and orange-yellow indicated a negative result. The reliability of the primers was verified based on fluorescence visualization RT-LAMP and electrophoresis band analysis.

[0035] (3) Validation of the specificity of RT-LAMP primers Electrophoresis and fluorescent dye detection confirmed that only the first set of primers could amplify the cDNA template from CVA-positive plants; the second and third sets of RT-LAMP primers failed to amplify the cDNA template from CVA-positive plants. Subsequent experiments were conducted using the first set of primers, amplifying the cDNA of five common sweet cherry viruses using RT-LAMP as templates to verify specificity. The results showed that the first set of primers produced a ladder-like band pattern in the CVA-containing samples, as shown in the image. Figure 2 As shown, no amplification was observed using other samples as templates and negative controls, indicating good primer specificity and no false positives. The SYBR Green I staining results were consistent with the electrophoresis results, further validating the specificity of the RT-LAMP primers. Therefore, the first set of primers (containing F3 / B3, FIP / BIP, and LB sequences) was selected for subsequent CVA specific detection primers.

[0036] Example 2 Establishment of a rapid RT-LAMP visualization detection method and optimization of the content of each component in the detection system 1. Initial reaction system of CVA RT-LAMP Following the NEB Bst 2.0 DNA polymerase instructions, the experimental conditions were optimized to establish a CVA RT-LAMP initial reaction system. The system consisted of 25 μL of the following components: 2.5 μL of 10× Thermopol reaction buffer, 8.0 μL of inner primers (FIP+BIP, 10 μmol / L), 1.0 μL of outer primers (F3+B3, 10 μmol / L), 1.0 μL of loop primers (LB, 10 μmol / L), 1.5 μL of MgSO4 (100 mM), 3.5 μL of dNTPs Mix (10 mM), 1.0 μL of Bst 2.0 DNA polymerase (8000 U / mL), 1.5 μL of template, and ddH2O to make up the volume. The reaction was amplified at 65℃ for 40 min in a PCR instrument.

[0037] To optimize the detection of Mg in the visualized RT-LAMP system 2+ The initial system was optimized using single-factor experiments, focusing on the concentration ratio of inner and outer primers, dNTPs, Bst 2.0, and the ratio of inner to outer primers. Betaine was introduced to reduce the risk of false positives. Variables included the ratio of inner to outer primer concentration, the concentration of the loop primer, and Mg. 2+ The concentrations of dNTPs, Bst 2.0 polymerase, and betaine were detailed in Table 2. After amplification, the optimal concentrations of each component were determined by the clarity of the ladder-like bands and fluorescence visualization results of agarose gel electrophoresis.

[0038] Table 2 Gradient settings of major components in the fluorescence visualization RT-LAMP detection system

[0039] 2. Optimization of dNTP content Based on the initial RT-LAMP reaction system, six dNTP concentrations (0 mmol / L, 0.5 mmol / L, 1.0 mmol / L, 1.5 mmol / L, 2.0 mmol / L, and 2.5 mmol / L) were set up for comparative experiments. The CVA detection results of the RT-LAMP reaction systems with different dNTP concentrations are as follows: Figure 3 As shown.

[0040] according to Figure 3 The results show that when the concentration of dNTPs is below 0.5 mmol·L⁻¹, -1 No amplification band was observed at that time; concentrations ranged from 1.0 to 2.5 mmol / L. -1Clear, stepped bands were produced with minimal variation. Fluorescence detection results (reaction tubes turned green) were consistent with electrophoresis results. Considering application requirements and future integration, 1.0 mmol·L⁻¹ was selected. -1 The optimal concentration of dNTPs.

[0041] 3. Optimization of betaine content Based on the optimized system described above, six betaine concentrations (0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mol / L) were used for comparative experiments. The CVA detection results for RT-LAMP reaction systems with different betaine contents are shown below. Figure 4 As shown.

[0042] according to Figure 4 The results show that clear bands were amplified at concentrations of 0.2 and 0.4 mol / L without the addition of betaine, with the brightest band at 0.4 mol / L. The bands were darker without betaine, and no amplified bands were observed at concentrations of 0.6–1.0 mol / L. The fluorescence detection results (positive tubes appear green) are consistent with the electrophoresis results. Considering that high concentrations of betaine would inhibit the reaction, and taking into account the application requirements, 0.2 mol / L was selected as the optimal betaine concentration.

[0043] 4.Mg 2+ Content optimization Based on the above optimized system, Mg was set in a gradient of 2 mmol / L within the range of 0–10 mmol / L. 2+ Comparative experiments were conducted to obtain different concentrations of Mg. 2+ The CVA detection results of the RT-LAMP reaction system are as follows: Figure 5 As shown.

[0044] according to Figure 5 The results show that clear bands were amplified at concentrations ranging from 2 to 10 mmol / L, with the clearest bands observed at 6 mmol / L. The fluorescence detection results (positive tubes appear green) are consistent with the electrophoresis results; no Mg was added. 2+ No amplification band was observed. Considering all application requirements, 6 mmol / L was selected as the optimal Mg concentration. 2+ concentration.

[0045] 5. Optimization of Bst2.0 DNA polymerase content Based on the optimized system described above, comparative experiments were conducted using a Bst 2.0 DNA polymerase concentration gradient (0.128–0.448 U / μL, with intervals of 0.064 U / μL). The CVA detection results for RT-LAMP reaction systems with different Bst 2.0 DNA polymerase concentrations are shown below. Figure 6 As shown.

[0046] according to Figure 6 The results show that clear bands can be amplified at concentrations ranging from 0.32 to 0.448 U / μL, with the clearest bands observed at 0.448 U / μL. The fluorescence detection results (positive tubes appear green) are consistent with the electrophoresis results. Considering cost and overall application requirements, 0.32 U / μL was selected as the optimal enzyme concentration.

[0047] 6. Optimization of inner and outer primer content and loop primer concentration Based on the above optimized system, the ratio of inner and outer primers and the concentration of the loop primer were optimized respectively. Comparative experiments were conducted with inner and outer primer ratios of 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1. The CVA detection results of the RT-LAMP reaction systems with different inner and outer primer ratios are shown below. Figure 7 As shown in A and B, comparative experiments were conducted with loop primer concentrations of 0.0 μmol / L, 0.4 μmol / L, 0.6 μmol / L, 0.8 μmol / L, 1.0 μmol / L, and 1.2 μmol / L. The CVA detection results for RT-LAMP reaction systems with different loop primer concentrations are shown in Figure 1. Figure 7 As shown in C and D.

[0048] according to Figure 7 The results show that ladder-like bands were amplified at all primer ratios, with the clearest and brightest bands observed at a 4:1 ratio. The fluorescence visualization results (positive tubes appear green) are consistent with the electrophoresis results. Loop primer concentrations were set from 0 to 1.2 μmol / L (in 0.2 μmol / L intervals). The comparison experiment showed that the bands were clearest at 0.4 μmol / L. The fluorescence visualization results (positive tubes appear green) are consistent with the electrophoresis results. Considering practical application requirements, a primer ratio of 4:1 and a loop primer concentration of 0.4 μmol / L were selected.

[0049] Example 3 Optimization of RT-LAMP reaction conditions Based on the optimized RT-LAMP system, different reaction temperatures and times were set for amplification using CVA cDNA as a template. After the reaction, 2.5 μL of the product was subjected to 1.5% agarose gel electrophoresis; 0.5 μL of 1000× SYBR Green I was added to the reaction tube to observe the color change. The optimal reaction temperature and time were finally determined based on the clarity of the gel bands and the fluorescence visualization results. Eight temperature gradients (58℃, 59℃, 60℃, 62℃, 65℃, 67℃, 69℃, and 70℃) were set for comparative experiments to optimize the reaction temperature. The CVA detection results of the RT-LAMP reaction system at different reaction temperatures are shown below. Figure 8As shown in A and B in the figure; six time gradients (25 min, 30 min, 35 min, 40 min, 45 min, and 50 min) were set to optimize the reaction time for comparative experiments, and the CVA detection results of the RT-LAMP reaction system with different reaction times were obtained as follows. Figure 8 As shown in C and D in the diagram.

[0050] according to Figure 8 The results show that when the reaction temperature is 60-70 ℃, the agarose gel electrophoresis results show waterfall-like bands, with the bands being the clearest at 62 ℃, and the RT-LAMP reaction solution all appearing green. Step-like bands are produced within a reaction time of 30-50 min. The step-like bands are brightest at 40 min, and the reaction solution is green (positive). Considering the application requirements, 62 ℃ is selected as the optimal reaction temperature, and 40 min as the optimal reaction time.

[0051] Example 4 RT-LAMP Sensitivity Detection Using the preserved CVA cDNA as a template, the concentration was determined to be 675.4 ng·μL using a micro-nucleic acid analyzer. -1 A 10-fold serial dilution was performed using DEPC-ddH2O to obtain 10 1 ~10 -6 A total of 7 concentrations were used. RT-PCR and RT-LAMP were performed using templates at each dilution. After the reaction, 2.5 μL of each amplification product was taken for 1.5% agarose gel electrophoresis and fluorescence visualization to compare the sensitivity of RT-LAMP and RT-PCR.

[0052] RT-LAMP assays were performed according to the optimized systems described in Examples 2 and 3. The primers for RT-PCR were CVA-6-F and CVA-6-R as shown in Table 1. Following the instructions for the Sangon Biotech 2X High-Fidelity PCR Mix premix, the reaction mixture consisted of: 25 μL of 2X High-Fidelity PCR Mix premix; 2.0 μL each of forward and reverse primers (10 μM); 1.5 μL of template DNA; and ddH2O to a final volume of 50 μL. The reaction program was 95℃ for 3 min, 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 15 s, for 35 cycles; followed by 72℃ for 5 min. After the reaction, 2.5 μL of the PCR product was subjected to 2% agarose gel electrophoresis for 15 min, and the bands were observed and photographed. The results of RT-LAMP and RT-PCR assays are shown below. Figure 9 As shown.

[0053] according to Figure 9 The results show that the detection sensitivity of RT-PCR is 675.4 × 10⁻⁶. -2 ng·µL-1 The detection sensitivity of RT-LAMP can reach 675.4 × 10⁻⁶. -4 ng·µL -1 This indicates that RT-LAMP is 100 times more sensitive than RT-PCR.

[0054] Example 5 RT-LAMP Specific Detection The specificity of the RT-LAMP system optimized in Examples 2 and 3 was verified. cDNA from six common sweet cherry viruses (CVA, PNRSV, PDV, CRLV, CGRMV, and LchV-1) preserved in the laboratory was used as templates for amplification (three replicates for each). Specificity was assessed by agarose gel electrophoresis and RT-LAMP visualization. The 202bp target band from the gel electrophoresis was further excised and sequenced. The accuracy of CVA RT-LAMP was verified by NCBI BLASTn alignment. The specificity of the RT-LAMP was obtained as follows: Figure 10 As shown.

[0055] according to Figure 10 The results show that only CVA-positive samples produced ladder-like bands (electrophoresis) and fluorescence signals (visualization). The visualization results of the amplified products were consistent with the gel electrophoresis results, with no cross-reactivity, indicating that the established CVA RT-LAMP detection method has good specificity. The target lane of the CVA was excised, and the amplified CVA product at approximately 202 bp was cloned and sequenced. The sequencing results were compared with NCBI BLASTn, and the results showed that the RT-LAMP amplified CVA sequence had 100%, 99.7%, and 99.9% sequence identity with the reported isolates Bd.P12 (GenBank accession number: OR148948.1), PAI3-5-3 (GenBank accession number: ON216678.1), and PVO8-25 (GenBank accession number: ON216675.1), respectively. This indicates that the established CVA RT-LAMP method has high accuracy.

[0056] Example 6 1. Development of RT-LAMP nucleic acid crude extraction and rapid detection technology To simplify the molecular detection of plant viruses, nucleic acid was extracted by crude lysis (approximately 15 min), and reverse transcriptase was added directly to the system (15 min) before visualization RT-LAMP detection (approximately 40 min). 0.5 μL of 10000×SYBRGreen I was pre-added to the tube cap, and the color change was observed after centrifugation with the tube closed.

[0057] Crude nucleic acid extraction (lysis-dilution method) includes the following steps: ① Prepare lysis buffer: 0.1M sodium citrate (pH 6.4) + 0.5% SDS + 1% β-mercaptoethanol (prepare fresh before use). ② Prepare neutralization buffer: 0.1M HCl. ③ Grinding: Take 20-50 mg of young leaves, cut them into small pieces, place them in a 2 mL EP tube containing steel beads, freeze quickly in liquid nitrogen, and then grind into powder. ④ Lysis: Add 500 μL of pre-cooled lysis buffer and mix by pipetting. ⑤ Heat denaturation: Incubate at 95℃ for 5 min. ⑥ Cooling: Let stand at room temperature for 2 min. ⑦ Neutralization: Add 25 μL of 0.1M HCl and mix by inverting. ⑧ Centrifugation: Centrifuge at 12,000 rpm for 5 min, and transfer 150 μL of supernatant to a new tube. ⑨ Dilution: Dilute the supernatant 1-6 times with DEPC-H2O to determine the suitable concentration.

[0058] 2. Confirmation of the dilution factor for the crude nucleic acid extract The supernatant (crude nucleic acid extract) diluted 1-6 times above was used as template for reverse transcription and RT-LAMP amplification of RNA in the same reaction system. The reaction system, in 25 μL volume, included the following components: 1 μL of 200 U / μL M-MLV Reverse Transcriptase, 4 μL of 5×M-MLV reaction buffer, 2.5 μL of 10×Thermopol reaction buffer, 4.0 μL of 10 μmol / L inner primer, 1.0 μL of 10 μmol / L outer primer, 1 μL of 10 μmol / L loop primer, 1.5 μL of 100 mM MgSO4 solution, 1 μL of 5 M betaine, 2.5 μL of 10 mM dNTPs Mix, 1.0 μL of 8000 U / mL Bst 2.0 DNA polymerase, 1.5 μL of template, and ddH2O to a final volume of 25 μL. The reaction conditions for reverse transcription of RNA are: first, treatment at 42℃ for 30 min, then treatment at 70℃ for 15 min; the reaction conditions for RT-LAMP amplification are: incubation at 60~70℃ for 30~50 min.

[0059] RT-LAMP detection results obtained by using supernatant at different dilution factors as reaction templates are as follows: Figure 13 As shown, according to Figure 13 The results show that the best fluorescence visualization results were obtained when the crude nucleic acid extract was diluted 2 to 4 times for LAMP reaction. However, the color development was not obvious below 1 fold, and it could not be effectively detected after being diluted 6 times. Therefore, the final dilution concentration of 2 to 4 times was determined to be optimal.

[0060] The final procedure for crude nucleic acid extraction from the test samples is as follows: Figure 11As shown in (1) to (4), (1) is to use a 2mL enzyme-free centrifuge tube with steel beads in advance, take sweet cherry leaves into the tube, freeze them with liquid nitrogen, and immediately crush the leaves into powder with a magnetic bead vibrator. Add 500μL of pre-cooled lysis buffer, mix by blowing, and cool at room temperature for 2 min after 5 min in a 95℃ water bath; (2) add 25μL of 0.1M HCl for neutralization, mix by inverting, and then centrifuge (centrifuge at 12000 rpm for 5 min); (3) take 150μL of supernatant into a new tube; (4) dilute the supernatant by 2 times with DEPC-H2O as a template.

[0061] 3. Validation of the RT-LAMP effect of crude nucleic acid extraction template Two μL of crude nucleic acid, diluted twofold, was used as an RT-LAMP template. The RT-LAMP of the crude template was performed following the steps described above, and the results were compared with those of the purified cDNA template to verify the feasibility of directly using the crude nucleic acid for RT-LAMP. The detection results are shown below. Figure 11 As shown.

[0062] according to Figure 11 The results show that the detection results of the crude RNA extract from CVA-infected sweet cherry samples were consistent with those of the cDNA template. Visualized RT-LAMP results were green for positive results and orange for negative results. This indicates that the crude nucleic acid extraction method can be used for RT-LAMP detection of CVA, thus shortening the nucleic acid extraction process to only 15 minutes. Based on this, RT-LAMP detection can meet the requirements for rapid detection of field samples, validating its applicability.

[0063] Example 7 Field sample detection using RT-LAMP technology Field samples were compared and detected using the CVA RT-LAMP system established in this invention and the conventional RT-PCR method. The CVA RT-LAMP system was performed using the rapid nucleic acid extraction technique (one-step method) as described in Example 6; the reaction system and procedure of the conventional RT-PCR method were the same as in Example 4.

[0064] Seventy sweet cherry leaf samples that had previously tested positive for CVA by ELISA were tested using RT-PCR and RT-LAMP to evaluate the feasibility of RT-LAMP for CVA detection. The distribution of the 70 sweet cherry leaf samples is shown in Table 3.

[0065] Table 3 Distribution of sweet cherry sample collection locations

[0066] The test results of 70 CVA-positive sweet cherry leaf samples are as follows: Figure 12 As shown. According to Figure 12 The results show that RT-LAMP detected 64 positive samples (detection rate 91.42%), which is significantly higher than RT-PCR's 50 positive samples (detection rate 71.42%), indicating that the established RT-LAMP method has higher detection accuracy than RT-PCR.

[0067] Conclusions and Discussion Cherry virus A (CVA) is one of the important latent viruses affecting sweet cherry production, and traditional monitoring methods are difficult. This study designed five sets of RT-LAMP primers based on the conserved region of the CVA coat protein (CP) gene. After screening and optimization, the optimal primer combination (CVA-1) and its optimal concentration ratio (FIP / BIP: 1.6 μmol·L⁻¹) were obtained. -1 F3 / B3: 0.4 μmol·L -1 LB: 0.2 μmol·L -1 The reaction system was further optimized (temperature 62℃, time 40 min). The method described in this invention achieves a detection limit (LOD) of 675.4 × 10⁻⁶ for CVAcDNA. -4 ng·μL -1 This invention demonstrates a sensitivity 100 times higher than conventional RT-PCR methods and exhibits no cross-reactivity with five common cherry viruses. Furthermore, the invention simplifies the nucleic acid extraction method, saving extraction time and offering advantages such as speed (40 min), low cost (isothermal amplification), and visualization. This invention is the first to establish a visual detection method for CVA virus based on RT-LAMP, and has been successfully applied to field samples, providing an effective tool for efficient monitoring of CVA in sweet cherries.

[0068] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An RT-LAMP primer set for detecting cherry virus A, characterized in that, The RT-LAMP primer set includes outer primer F3 as shown in SEQ ID NO.1, outer primer B3 as shown in SEQ ID NO.2, inner primer FIP as shown in SEQ ID NO.3, and inner primer BIP as shown in SEQ ID NO.

4.

2. The RT-LAMP primer set according to claim 1, characterized in that, The RT-LAMP primer set also includes the circular primer LB as shown in SEQ ID NO.

5.

3. A reagent or kit for detecting cherry virus A, characterized in that, Includes the RT-LAMP primer set as described in claim 1 or 2.

4. The application of the RT-LAMP primer set according to claim 1 or 2 or the reagent or kit according to claim 3 in the cultivation of virus-free seedlings and / or in seedling quarantine.

5. The application according to claim 4, characterized in that, The seedling quarantine includes testing for cherry virus A.

6. A method for detecting cherry virus A using RT-LAMP, characterized in that, Includes the following steps: 1) Extract total RNA from the sample to be tested; reverse transcribe the total RNA to obtain cDNA; 2) Using the cDNA as a template, perform RT-LAMP amplification using the RT-LAMP primer set described in claim 1 or 2. If a specific amplification product is detected, determine that the sample to be tested contains cherry virus A; or, include the following steps: (1) Extract total RNA from the sample to be tested; (2) Using the total RNA as a template, reverse transcription and RT-LAMP amplification of RNA are performed sequentially in the same reaction system. The RT-LAMP amplification is performed using the RT-LAMP primer set described in claim 1 or 2. If a specific amplification product is detected, it is determined that the sample to be tested contains cherry virus A.

7. The method according to claim 6, characterized in that, The reaction conditions for RT-LAMP amplification in step 2) are incubation at 60-70℃ for 30-50 min. The RT-LAMP amplification reaction system in step 2) is 25 μL and includes the following components: 2.5 μL of 10×Thermopol reaction buffer, 4.0 μL of 10 μmol / L inner primer, 1.0 μL of 10 μmol / L outer primer, 1 μL of 10 μmol / L loop primer, 1.5 μL of 100 mM MgSO4 solution, 1 μL of 5M betaine, 2.5 μL of 10 mM dNTPs Mix, 1.0 μL of 8000 U / mL Bst 2.0 DNA polymerase, 1.5 μL of template, and ddH2O to a final volume of 25 μL.

8. The method according to claim 6, characterized in that, The reaction system in step (2) is 25 μL and includes the following components: 1 μL of 200 U / μL M-MLV Reverse Transcriptase, 4 μL of 5×M-MLV reaction buffer, 2.5 μL of 10×Thermopol reaction buffer, 4.0 μL of 10 μmol / L inner primer, 1.0 μL of 10 μmol / L outer primer, 1 μL of 10 μmol / L loop primer, 1.5 μL of 100 mM MgSO4 solution, 1 μL of 5M betaine, 2.5 μL of 10 mM dNTPs Mix, 1.0 μL of 8000 U / mL Bst 2.0 DNA polymerase, 1.5 μL of template, and ddH2O to bring the total to 25 μL.

9. The method according to claim 6, characterized in that, The reaction conditions for reverse transcription of RNA in step (2) are: first, treatment at 42℃ for 15 min, and then treatment at 90℃ for 3 min. The reaction conditions for RT-LAMP amplification in step (2) are 60~70℃ for 30~50 min.

10. The method according to any one of claims 6 to 8, characterized in that, The detection methods for the specific amplification products include electrophoresis detection and / or fluorescent dye colorimetric detection; If the electrophoresis results show waterfall-like trapezoidal characteristic bands, it is determined that the sample contains cherry virus A. If the result of the fluorescent dye colorimetric detection is fluorescence, then it is determined that the sample contains cherry virus A. The fluorescent dye includes SYBR Green I.

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