CN116891911B - Method for detecting tomato brown wrinkle fruit virus based on RT-RAA and lateral flow chromatography test strip - Google Patents
Method for detecting tomato brown wrinkle fruit virus based on RT-RAA and lateral flow chromatography test strip Download PDFInfo
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Abstract
The invention discloses a method for detecting tomato brown wrinkle fruit virus based on RT-RAA and a lateral flow chromatography test strip. The invention provides an RT-RAA primer and probe combination for detecting tomato brown wrinkle fruit virus, and also provides a method for detecting tomato brown wrinkle fruit virus by combining a lateral flow chromatography test strip by utilizing the primer and probe combination. The method of the invention can realize the rapid detection of the tomato brown wrinkle fruit virus, and has low requirements on equipment, high specificity and sensitivity, accurate detection result and difficult occurrence of false positive. The invention is helpful for rapid detection of tomato brown wrinkle fruit virus and control of tomato brown wrinkle fruit virus.
Description
Technical Field
The invention belongs to the technical field of plant virus detection. More particularly, it relates to a method for detecting tomato brown wrinkle fruit virus based on RT-RAA and a lateral flow chromatography test strip.
Background
Tomato brown wrinkle virus (Tomato brown rugose fruit virus, toBRFV) is a new positive single-strand RNA (+ssRNA) virus in recent years, belongs to the family of the broom virus (VIRGAVIRIDAE), tobacco mosaic virus (Tobamovirus), genome full length 6.2-6.4 kb. ToBRFV are tomato (Solanum lycopersicum) and Capsicum annuum (Capsicum annuum), the flowers and fruits of the infected plants are greatly reduced, the fruits are deformed, and the fruits are deformed, browned and necrotic when serious, so that the eating value and the yield are seriously affected. In 2015, the virus rapidly spreads worldwide, is detected in more than 30 countries or regions, spans europe, america, africa and asia, and has the characteristics of seed virus spreading, fast spreading speed and high detection rate. Rapid and accurate detection of ToBRFV is critical to preventing its propagation.
Since ToBRFV is susceptible to infection by one or more viruses of the same genus simultaneously with the infection by the same strain. Thus, higher specificity is required for the detection of ToBRFV. In the existing ToBRFV detection technology, the Enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) has no good specificity; the existing ToBRFV commercial ELISA detection kit has cross reaction with tomato mosaic virus (Tomato mosaic virus, toMV) and tobacco mosaic virus (Tobacco mosaic virus, TMV); high throughput sequencing technology (Next-generation sequencing, NGS) is limited by laboratory conditions and detection costs and cannot be used for routine rapid detection; although the common RT-PCR and the real-time fluorescence RT-PCR have the advantages of high sensitivity and short detection time, professional technicians with certain experience are required to finish the operation in a laboratory, the requirements on equipment are high, and the defects of complicated operation, long time consumption, high cost and the like are overcome. Zhiyong et al (2021) developed a specific test strip using ToBRFV particles as antigen, which can rapidly detect ToBRFV, but has lower detection sensitivity, 32 times lower than that of ordinary RT-PCR. There is a need to provide a product and method that can detect ToBRFV simply, quickly, and accurately.
The recombinase-mediated isothermal nucleic acid amplification (recombinase-aided amplification, RAA) technology is an in-vitro nucleic acid isothermal amplification method (Ma Wen, etc., 2021) imitating T4 phage, can realize DNA exponential amplification (Jiao et al., 2019) within 20min under the condition of constant temperature without a high-cost temperature-raising instrument, is simple and convenient to operate, has quick and sensitive reaction, and is suitable for rapid detection of viral genome DNA or RNA (Li et al., 2019). However, compared with PCR, RAA has shorter time, large difficulty in designing primers and probes, difficult construction of the method and few mature application cases in the field of plant disease detection. Compared with the traditional agarose gel electrophoresis detection, the lateral flow chromatography test strip (lateral flow assay, LFA) adopts a chromatography double-antibody sandwich method to rapidly detect the nucleic acid amplification product (Fu et al, 2014), and the method is simple to operate, rapid in interpretation, free of toxic substances, free of any instrument and equipment (Aktas et al, 2019), convenient for realizing the on-site rapid detection of viruses, but incapable of independently realizing the accurate detection of ToBRFV.
Disclosure of Invention
The invention aims to solve the technical problem of how to overcome the defects and the shortcomings of the prior art by utilizing the advantages of two technologies of isothermal nucleic acid amplification and lateral flow chromatography test paper strip mediated by recombinant enzyme, and provides a method for detecting tomato brown wrinkle fruit virus based on RT-RAA and lateral flow chromatography test paper strip.
It is a first object of the present invention to provide an RT-RAA primer and probe combination for detection of tomato brown wrinkle fruit virus.
A second object of the invention is to provide the use of said RT-RAA primer and probe combination for detecting tomato brown wrinkle virus.
A third object of the present invention is to provide the use of the RT-RAA primer and probe combination for the preparation of a product for detecting tomato brown wrinkle fruit virus.
A fourth object of the invention is to provide a kit for detecting tomato brown wrinkle virus.
It is a fifth object of the present invention to provide a method for detecting tomato brown wrinkle virus based on RT-RAA.
The sixth object of the invention is to provide a method for detecting tomato brown wrinkle virus based on RT-RAA and a lateral flow chromatography test strip.
The above object of the present invention is achieved by the following technical scheme:
According to the invention, a plurality of RT-RAA primers and probes are respectively designed based on a plurality of different gene sequences of the tomato brown wrinkle virus, and through continuous cyclic verification, adjustment and re-verification processes, RT-RAA primers and probes with relatively good effects are obtained based on CP gene design, and RT-RAA primer and probe combinations for detecting the tomato brown wrinkle virus and corresponding detection methods are obtained through further screening and optimizing the primer and probe combinations, so that the specificity, sensitivity and rapid detection of the tomato brown wrinkle virus can be realized.
The invention provides an RT-RAA primer and probe combination for detecting tomato brown wrinkle virus, which comprises a primer ToB-RA5, a primer ToB-RA6 and a probe LF-P1; the sequence of the primer ToB-RA5 is shown as SEQ ID NO. 1; the sequence of the primer ToB-RA6 is shown as SEQ ID NO. 2; the sequence of the probe LF-P1 is shown as SEQ ID NO. 3.
By combining the RT-RAA primer and the probe and combining a lateral flow chromatography test strip, the detection of the tomato brown wrinkle fruit virus can be realized.
In order to facilitate detection by combining with a flow-measuring chromatographic test strip, optionally, biotin (Biotin) is marked at the 5' end of the ToB-RA6 sequence of the primer; THE 5 'end of THE probe LF-P1 sequence is marked with a fluorescent group FAM, THE 31 st base is marked with tetrahydrofuran residue (THE), and THE 3' end is modified by C3 Spacer.
The RT-RAA primer and probe combination can be used for realizing the specific detection of the tomato brown wrinkle fruit virus. Therefore, the invention also claims the use of said RT-RAA primer and probe combination for detecting tomato brown wrinkle virus.
The invention also claims the application of the RT-RAA primer and probe combination in preparing a product for detecting tomato brown wrinkle fruit virus.
The invention also provides a kit for detecting the tomato brown wrinkle fruit virus, which comprises the RT-RAA primer and the probe combination.
Specifically, the kit also contains reagents required by RT-RAA reaction.
Specifically, the kit also contains 10 mu mol/L magnesium acetate.
As an alternative embodiment, the kit further comprises a lateral flow chromatography test strip.
Specifically, the lateral flow chromatography test strip is a test strip matched with a marker on the primer and/or the probe, and can be used for detecting amplification products of the primer and the probe.
Optionally, the lateral flow chromatography test strip is a lateral flow chromatography test strip marked with colloidal gold.
The invention also provides a method for detecting tomato brown wrinkle virus, which comprises the following steps:
S1, extracting total RNA of a sample to be detected;
S2, taking the total RNA obtained in the S1 as a template, and carrying out RT-RAA reaction by using the primer and probe combination;
S3, detecting the amplified product by agarose gel electrophoresis; if the specific band with the size of 200bp appears, the detection result is positive, otherwise, the detection result is negative.
The invention also provides a method for detecting tomato brown wrinkle fruit virus based on RT-RAA and a lateral flow chromatography test strip, which comprises the following steps:
S1, extracting total RNA of a sample to be detected;
S2, taking the total RNA obtained in the S1 as a template, and carrying out RT-RAA reaction by using the primer and probe combination;
S3, diluting the amplified product, and detecting by using a lateral flow chromatography test strip; if the strips appear at the quality control line and the detection line of the test strip, the detection result is positive; if the strip appears at the quality control line of the test strip, the detection result is negative; if no strip appears at the quality control line and the detection line of the test strip, the detection result is invalid.
The amplified product is diluted and then detected by a lateral flow chromatography test strip, so that the rapid detection on site can be realized. Specifically, the lateral flow chromatography test strip is a lateral flow chromatography test strip marked with colloidal gold.
Specifically, in the method, the concentration of the primer in a reaction system used in the RT-RAA reaction is 0.12-0.4 mu mol/L; the concentration of the probe ranges from 0.06 to 0.2 mu mol/L.
More specifically, the concentration of the primer was 0.216. Mu. Mol/L.
More specifically, the concentration of the probe was 0.06. Mu. Mol/L.
Specifically, in the above method, the reaction conditions used for the RT-RAA reaction are: and (3) performing constant temperature treatment at 42 ℃ for 10-15 min.
More specifically, the treatment time was 15min.
The invention has the following beneficial effects:
The invention provides an RT-RAA primer and probe combination for detecting tomato brown wrinkle fruit virus, and also provides a method for detecting tomato brown wrinkle fruit virus by combining a lateral flow chromatography test strip by utilizing the primer and probe combination. The method of the invention can realize the rapid detection of the tomato brown wrinkle fruit virus, and has low requirements on equipment, high specificity and sensitivity, accurate detection result and difficult occurrence of false positive. The invention is helpful for rapid detection of tomato brown wrinkle fruit virus and control of tomato brown wrinkle fruit virus.
Drawings
FIG. 1 shows the results of screening RT-RAA primers and probes; the positive samples of ToBRFV are detected by 1, 3, 5 and 7 in the figure, and the blank controls are detected by 2, 4, 6 and 8 in the figure; in the figures, primer and probe combinations corresponding to 1-8 are ：ToB-RA3/ToB-RA4/LF-P1、ToB-RA3/ToB-RA4/LF-P1、ToB-RA3/ToB-RA4/LF-P2、ToB-RA3/ToB-RA4/LF-P2、ToB-RA5/ToB-RA6/LF-P1、ToB-RA5/ToB-RA6/LF-P1、ToB-RA5/ToB-RA6/LF-P2 and ToB-RA5/ToB-RA6/LF-P2 in sequence.
FIG. 2 shows the results of optimization of probe concentration in the RT-RAA-LFA detection method; the positive samples of ToBRFV are detected by 1,3, 5 and 7 in the figure, and the blank controls are detected by 2,4, 6 and 8 in the figure; in the figure, the probe concentrations corresponding to 1 to 8 were 0.06. Mu. Mol/L, 0.1. Mu. Mol/L, 0.2. Mu. Mol/L, 0.4. Mu. Mol/L, and 0.4. Mu. Mol/L, in this order.
FIG. 3 shows the results of specific detection by RT-RAA detection method; 1 in the figure is ToBRFV RNA samples; 2 in the figure is a TMV RNA sample; 3 in the figure are ToMV RNA samples; 4 in the figure is ToMMV RNA samples; FIG. 5 shows PMMoV RNA samples; FIG. 6 shows PepMV RNA samples; 7 in the figure is ToRSV RNA samples; the figure 8 shows a TRSV RNA sample.
FIG. 4 shows the results of specific detection by RT-RAA-LFA detection method; 1 in the figure is ToBRFV RNA samples; 2 in the figure is a TMV RNA sample; 3 in the figure are ToMV RNA samples; 4 in the figure is ToMMV RNA samples; FIG. 5 shows PMMoV RNA samples; FIG. 6 shows PepMV RNA samples; 7 in the figure is ToRSV RNA samples; 8in the figure is a TRSV RNA sample; 9 in the figure is a blank; 10 in the figure is a healthy tomato leaf.
FIG. 5 shows the sensitivity test results of the conventional RT-PCR method; m is M DL2000 Marker; the concentration of the RNA samples corresponding to 1 to 10 in the figure is in order 92.4、9.24×100、9.24×10-1、9.24×10-2、9.24×10-3、9.24×10-4、9.24×10-5、9.24×10-6、9.24×10-7、9.24×10-8ng/μL.
FIG. 6 shows the sensitivity test results of the RT-RAA-LFA detection method; the concentration of the RNA samples corresponding to 1 to 10 in the figure is in order 92.4、9.24×100、9.24×10-1、9.24×10-2、9.24×10-3、9.24×10-4、9.24×10-5、9.24×10-6、9.24×10-7、9.24×10-8ng/μL.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The KIT for carrying out the RT-RAA reaction is an RNA isothermal rapid amplification KIT, and is purchased from Anpu future (Changzhou) biotechnology Co., ltd, and the product number is WLRN 8239 KIT.
The lateral flow chromatography test strip used in the invention is purchased from Anpu future (Changzhou) biotechnology limited company, and the product number is WLFS 8234.
Example 1 design of primers and probes and establishment of detection method
1. Design of primers and probes
According to the invention, a plurality of RT-RAA primers and probes are respectively designed based on ToBRFV different gene sequences, and RT-RAA primers and probes with relatively good effects are obtained based on CP gene design through continuous cyclic verification, adjustment and re-verification processes, wherein the RT-RAA primers and probes comprise primers ToB-RA3, toB-RA4, toB-RA5 and ToB-RA6, and probes LF-P1 and LF-P2, and an RT-RAA and RT-RAA-LFA detection method for detecting ToBRFV is constructed based on the primers. In order to facilitate the comparison of the detection effect, the invention also utilizes common RT-PCR primers ToBRFV-F-5506 and ToBRFV-R-6186 in the construction of the method for detecting ToBRFV. The sequences of the primers and probes and the modifications carried by the primers and probes are shown in Table 1:
TABLE 1 primer and probe sequences
| Name of the name | Primer sequence and its carrying mark |
| ToBRFV-F-5506 | 5'-GTCCCGATGTCTGTAAGGCTTGC-3' |
| ToBRFV-R-6186 | 5'-GCAGGTGCAGAGGACCATTGTAA-3' |
| ToB-RA3 | 5'-CACTAGGTAATCAGTTCCAAACACAACAAG-3' |
| ToB-RA4 | 5'-Biotin-TAGAGGATCTAGTACCGCATTGTACCTATAC-3' |
| ToB-RA5 | 5'-CTAATTCACTAGGTAATCAGTTCCAAACACAAC-3' |
| ToB-RA6 | 5'-Biotin-TATAATCCTATTTCTAGTATCGAAAGCTCCTAA-3' |
| LF-P1 | 5'-FAM-CTAGAACAACCGTTCAACGGCAATTTAGCG(THF)AGTGTGGAAACCTGTC-C3-space3' |
| LF-P2 | 5'-TCAACGGCAATTTAGCGAAGTGTGGAAACC(THF)GTCCCTCAAGTCACT-C3-space3' |
Note that: the primer labeled with Biotin (Biotin) at the 5' end is a downstream primer.
2. ToBRFV RT-RAA and RT-RAA-LFA detection method
(1) Extraction of viral RNA
Extracting total RNA of infected ToBRFV tomato leaves and healthy tomato leaves respectively according to the instruction of commercial plant total RNA extraction kit, detecting RNA concentration with an ultra-micro spectrophotometer, and preserving at-80deg.C for use.
(2) ToBRFV RT-RAA and RT-RAA-LFA detection
And (3) carrying out RT-RAA-LFA detection by taking extracted total RNA infected with ToBRFV tomato leaves as a template, wherein a KIT used in the RT-RAA reaction is an RNA isothermal rapid amplification KIT (product number is WLRN8209 KIT). The volume of the RT-RAA-LFA detection system is 50 mu L, wherein Buffer A29.4 mu L,10 mu mol/L upstream primer 2 mu L,10 mu mol/L downstream primer 1.8 mu L,10 mu mol/L probe 0.5 mu L, total RNA 2 mu L, buffer B (10 mu mol/L magnesium acetate) 2.5 mu L and double distilled water make up 50 mu L; the reaction tube is turned upside down for 8-10 times, fully and evenly mixed, and after oscillating and centrifuging, the reaction tube is transferred into a constant temperature heating device for heating for 15min at 42 ℃. When RT-RAA is used for detection, agarose gel electrophoresis is used for detecting the amplified product; if the specific band with the size of 200bp appears, the detection result is positive, otherwise, the detection result is negative. When the RT-RAA-LFA is used for detection, 10 mu L of the RT-RAA-LFA is taken out after heating and added into a 1.5mL centrifuge tube containing 190 mu L of deionized water, the RT-RAA-LFA is uniformly mixed, 50 mu L of diluted reaction product is sucked, and the reaction product is dripped into a sample-adding hole of a lateral flow chromatography test strip, and the change condition of the test strip is observed after 5min. If 2 strips appear on the test strip (1 blue strip appears at the quality control line and 1 red strip appears at the detection line), the detection result is positive; if 1 blue strip appears at the quality control line of the test strip, the detection result is negative; if no strip appears at the quality control line and the detection line of the test strip, the detection result is invalid.
3. Screening of RT-RAA-LFA detection primer probes
The RT-RAA primers and probes shown in the table 1 are respectively combined, screened and optimized, 4 combinations are respectively ToB-RA3/ToB-RA4/LF-P1, toB-RA3/ToB-RA4/LF-P2, toB-RA5/ToB-RA6/LF-P1 and ToB-RA5/ToB-RA6/LF-P2, other conditions are kept unchanged, the combination systems are respectively detected according to the method, double distilled water is used as a blank control, and the change condition of the colloidal gold marked flow measurement chromatographic test strips under different combinations is observed.
The results of the RT-RAA-LFA detection primer probe screening are shown in FIG. 1. As can be seen from FIG. 1, when the 4 primer probes are combined, the ToBRFV colloidal gold test strip detection lines all have obvious red bands, and the blank control has no red bands. The detection line of the combined ToB-RA3/ToB-RA4/LF-P1ToBRFV colloidal gold test strip has the weakest intensity, the detection line of the combined ToB-RA3/ToB-RA4/LF-P2 has the inferior intensity, the detection lines of the combined ToB-RA5/ToB-RA6/LF-P1 and the combined ToB-RA5/ToB-RA6/LF-P2 have the strongest intensities, the intensities of the two detection lines are basically consistent, but the time for the occurrence of an obvious detection line of the ToB-RA5/ToB-RA6/LF-P1 combination is shorter, and the subsequent experiment selects the ToB-RA5/ToB-RA6/LF-P1 combination.
4. Optimization of RT-RAA-LFA detection method
The invention optimizes the concentration of the probe in the RT-RAA-LFA detection method. Specifically, in the 50. Mu.L reaction system, the final probe concentrations were set to 0.06, 0.1, 0.2 and 0.4. Mu. Mol/L, respectively, and the other conditions were kept unchanged. According to the method, the optimization system is detected respectively, double distilled water is used as a blank control, and the change condition of the colloidal gold-marked flow measurement chromatographic test strip under different combinations is observed.
The results of the optimization of probe concentration in the RT-RAA-LFA detection method are shown in FIG. 2. As can be seen from FIG. 2, when the probe concentrations were 0.06, 0.1, 0.2, and 0.4. Mu. Mol/L, respectively, the detection lines of the colloidal gold-labeled lateral flow chromatography test strips all appeared as distinct red bands, and there was no distinct difference between the detection results of the 3 samples. However, when the probe concentration was too high (0.4. Mu. Mol/L), the blank was false positive (8 in FIG. 2), so 0.06. Mu. Mol/L was selected as the optimal concentration of probe in the RT-RAA-LFA detection system.
Example 2 specific assay
The tested toxin sources and plants used for specific detection comprise: toBRFV, tobacco mosaic virus (Tobacco mosaic virus, TMV), tomato mosaic virus (Tomato mosaic virus, toMV), tomato mottle mosaic virus (Tomato mottle mosaic virus, toMMV), pepper mottle virus (PEPPER MILD mottle virus, PMMoV), pepsin mosaic virus (Pepino mosaic virus, pepMV), tomato ringspot virus (Tomato ringspot virus, toRSV), tobacco ringspot virus (Tobacco ringspot virus, TRSV), a total of 8 viral RNA samples, and further including healthy tomato and pepper leaf samples, all of which were maintained in the inventor laboratory.
And respectively carrying out specificity detection on the 8 virus RNA samples and the healthy tomato leaf RNA by using the optimized RT-RAA and RT-RAA-LFA detection methods, setting 3 repetitions for each sample by taking double distilled water as a blank control, and judging whether the established detection method has specificity or not according to the detection result.
The results of the specific assay of the RT-RAA assay are shown in FIG. 3. The specific detection result of the RT-RAA-LFA detection method is shown in FIG. 4. As can be seen from FIG. 3, the primers and probes of the present invention can amplify ToBRFV efficiently, the size of the target fragment is 200bp, and the primers and probes have no specific amplification to other viruses. As can be seen from FIG. 4, only the colloidal gold strip of ToBRFV positive samples detected a distinct red band, while other viral RNAs only showed one blue control line. The results show that the RT-RAA primer and probe combination can only specifically amplify ToBRFV, and the RT-RAA and RT-RAA-LFA detection methods disclosed by the invention have good specificity and can specifically and accurately detect ToBRFV.
Example 3 sensitivity detection
Detection of different concentrations ToBRFV RNA: performing ultra-micro spectrophotometry detection on total RNA of ToBRFV infected tomato leaves, measuring the concentration of the total RNA to be 92.4 ng/. Mu.L, performing 10-time gradient dilution on the RNA solution, sequentially obtaining RNA solutions with the concentration of 92.4、9.24×100、9.24×10-1、9.24×10-2、9.24×10-3、9.24×10-4、9.24×10-5、9.24×10-6、9.24×10-7、9.24×10-8ng/μL, respectively performing RT-RAA-LFA detection and common RT-PCR detection by taking RNA solutions with different concentrations as templates, and comparing the sensitivity of 2 methods.
The common RT-PCR detection method comprises the following steps: the primer ToBRFV-F-5506/ToBRFV-R-6186 is used for carrying out one-step RT-PCR reaction (TAKARA, beijing) on the extracted total RNA of the tomato leaf infected with ToBRFV, and the reaction system is as follows: 2 XOne Step Buffer 12.5. Mu.L, 1.0. Mu.L each of the upstream and downstream primers (10. Mu.M), PRIMESCRIPT ONE STEP ENZYME MIX 1.0.0. Mu.L, total RNA 1.5. Mu.L, RNASE FREE DDH 2 O up to 25. Mu.L. The reaction procedure: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 50s,35 cycles; extending at 72 ℃ for 10min; mu.L of the PCR reaction solution was subjected to electrophoresis (120V, 30 min) using 1.5% agarose gel, and the detection result was analyzed by a gel imaging system.
The detection sensitivity results of the conventional RT-PCR detection method are shown in FIG. 5. The sensitivity test results of the RT-RAA-LFA test method are shown in FIG. 6. As can be seen from FIGS. 5 and 6, the sensitivity was 9.24X10. 10 -4 ng/. Mu.L (FIG. 5) when ToBRFV RNA was detected by the conventional RT-PCR method; when ToBRFV RNA is detected by using the RT-RAA-LFA method, the detection line of the colloidal gold test strip gradually weakens when the concentration of RNA is 9.24 multiplied by 10 -4 ng/. Mu.L, but when the concentration is 9.24 multiplied by 10 -6 ng/. Mu.L, the detection line can still be generated by using the colloidal gold test strip, which shows that the sensitivity of the detection by using the RT-RAA-LFA method is 9.24 multiplied by 10 -6 ng/. Mu.L (figure 6), and is improved by about 10 times compared with that of the common RT-PCR method.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. An RT-RAA primer and probe combination for detecting tomato brown wrinkle virus, which is characterized by comprising a primer ToB-RA5, a primer ToB-RA6 and a probe LF-P1; the sequence of the primer ToB-RA5 is shown as SEQ ID NO. 1; the sequence of the primer ToB-RA6 is shown as SEQ ID NO. 2; the sequence of the probe LF-P1 is shown in SEQ ID NO. 3; the 5' end of the primer ToB-RA6 sequence is marked with biotin; the 5 'end of the probe LF-P1 sequence is marked with a fluorescent group FAM, the 31 st base is marked with tetrahydrofuran residue, and the 3' end is modified by C3 Spacer.
2. Use of the RT-RAA primer and probe combination according to claim 1 for detection of tomato brown wrinkle fruit virus.
3. Use of the RT-RAA primer and probe combination according to claim 1 for the preparation of a product for detection of tomato brown wrinkle fruit virus.
4. A kit for detecting tomato brown wrinkle virus, comprising the RT-RAA primer and probe combination of claim 1.
5. The kit according to claim 4, wherein the kit further comprises reagents required for the RT-RAA reaction.
6. The kit of claim 5, further comprising a lateral flow chromatographic test strip.
7. A method for detecting tomato brown wrinkle virus based on RT-RAA, which is characterized by comprising the following steps:
S1, extracting total RNA of a sample to be detected;
s2, carrying out RT-RAA reaction by using the total RNA obtained in the S1 as a template and the primer and probe combination of claim 1;
s3, detecting the amplified product by agarose gel electrophoresis; if the specific band with the size of 200 bp appears, the detection result is positive, otherwise, the detection result is negative.
8. A method for detecting tomato brown wrinkle virus based on RT-RAA and a lateral flow chromatography test strip, which is characterized by comprising the following steps:
S1, extracting total RNA of a sample to be detected;
s2, carrying out RT-RAA reaction by using the total RNA obtained in the S1 as a template and the primer and probe combination of claim 1;
S3, diluting the amplified product, and detecting by using a lateral flow chromatography test strip; if the strips appear at the quality control line and the detection line of the test strip, the detection result is positive; if the strip appears at the quality control line of the test strip, the detection result is negative; if no strip appears at the quality control line and the detection line of the test strip, the detection result is invalid.
9. The method according to claim 8, wherein the concentration of the primer in the reaction system used in the RT-RAA reaction is 0.12 to 0.4. Mu. Mol/L; the concentration of the probe ranges from 0.06 to 0.2 mu mol/L.
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