CN116891911A - 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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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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 recently developed positive single-stranded RNA (+ssRNA) virus belonging to the family of the broom viruses (Virgaviridae), the genus tobamoviruses (Tobamoviruses), the genome being 6.2-6.4 kb in length. The main hosts of ToBRFV are tomato (Solanum lycopersicum) and Capsicum annuum, flowers and fruits of infected plants are greatly reduced, fruits are deformed, and fruit deformity, browning and necrosis are caused when serious, so that the edible 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. The rapid and accurate detection of ToBRFV is a key to preventing its transmission.
Since ToBRFV is susceptible to infection by one or more viruses of the same genus at the same time. Thus, higher specificity is required for detection against ToBRFV. In the existing detection technology of ToBRFV, an 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 a lower detection sensitivity, 32 times lower than that of ordinary RT-PCR. There is a need to provide a product and method for detecting ToBRFV that is simple, rapid and accurate.
The recombinase-mediated isothermal nucleic acid amplification (RAA) technology is an in vitro nucleic acid isothermal amplification method (Ma Wen et al, 2021) imitating T4 phage, can realize DNA exponential amplification (Jiao et al, 2019) under the condition of constant temperature within 20min without a high-cost temperature-raising instrument, is simple and convenient to operate, has rapid 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 nucleic acid amplification products (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 achieving on-site rapid detection of viruses, but incapable of independently achieving 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 detection of 1, 3, 5 and 7 in the figure is ToBRFV positive sample, and the detection of 2, 4, 6 and 8 in the figure is blank control; in the figures, the primer and probe combinations corresponding to 1-8 are as follows: toB-RA3/ToB-RA4/LF-P1, toB-RA3/ToB-RA4/LF-P2, toB-RA5/ToB-RA6/LF-P1, toB-RA5/ToB-RA6/LF-P2, and ToB-RA5/ToB-RA6/LF-P2.
FIG. 2 shows the results of optimization of probe concentration in the RT-RAA-LFA detection method; the detection of 1, 3, 5 and 7 in the figure is ToBRFV positive sample, and the detection of 2, 4, 6 and 8 in the figure is blank control; in the figure, the probe concentrations corresponding to 1to 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; FIG. 1 shows ToBRFV RNA samples; 2 in the figure is a TMV RNA sample; 3 in the figure are ToMV RNA samples; 4 in the figure are ToMMV RNA samples; FIG. 5 shows PMMoV RNA samples; FIG. 6 shows PepMV RNA samples; 7 in the figure is ToRSV RNA sample; the figure 8 shows a TRSV RNA sample.
FIG. 4 shows the results of specific detection by RT-RAA-LFA detection method; FIG. 1 shows ToBRFV RNA samples; 2 in the figure is a TMV RNA sample; 3 in the figure are ToMV RNA samples; 4 in the figure are ToMMV RNA samples; FIG. 5 shows PMMoV RNA samples; FIG. 6 shows PepMV RNA samples; 7 in the figure is ToRSV RNA sample; 8 in 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 1to 10 in the figure was 92.4 and 9.24X10 in this order 0 、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 -8 ng/μL。
FIG. 6 shows the sensitivity test results of the RT-RAA-LFA detection method; the concentration of the RNA samples corresponding to 1to 10 in the figure was 92.4 and 9.24X10 in this order 0 、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 -8 ng/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 limited company, and the product number is WLRN8209KIT.
The lateral flow chromatography test strip used in the invention is purchased from Anpu future (Changzhou) biotechnology limited company, and has the product number WLFS8204.
Example 1 design of primers and probes and establishment of detection method
1. Design of primers and probes
According to the method, a plurality of RT-RAA primers and probes are respectively designed based on a plurality of different gene sequences of ToBRFV, 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 comparison of detection effects, the invention also utilizes common RT-PCR primers ToBRFV-F-5506 and ToBRFV-R-6186 in constructing a 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. RT-RAA and RT-RAA-LFA detection method of ToBRFV
(1) Extraction of viral RNA
Total RNA of tomato leaves infected with ToBRFV and healthy tomato leaves is extracted respectively according to the instruction of commercial plant total RNA extraction kit, and the RNA concentration is detected by an ultra-micro spectrophotometer and stored at-80 ℃ for standby.
(2) RT-RAA and RT-RAA-LFA detection of ToBRFV
And (3) carrying out RT-RAA-LFA detection by taking extracted total RNA of tomato leaves infected with ToBRFV as a template, wherein a KIT used in the RT-RAA reaction is an RNA isothermal rapid amplification KIT (with the product number of 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 show obvious red bands, and the blank control has no red bands. The ToB-RA3/ToB-RA4/LF-P1 combined ToBRFV colloidal gold test strip has the weakest detection line, the ToB-RA3/ToB-RA4/LF-P2 combined detection line has the inferior intensity, the ToB-RA5/ToB-RA6/LF-P1 combined detection line and the ToB-RA5/ToB-RA6/LF-P2 combined detection line have the strongest intensities, the intensities are basically consistent, but the time for the ToB-RA5/ToB-RA6/LF-P1 combined detection line to appear obvious detection line is shorter, and the ToB-RA5/ToB-RA6/LF-P1 combination is selected in the subsequent experiments.
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), 8 total viral RNA samples, and healthy tomato and pepper leaf samples, all of which were maintained in the inventor's 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 shown in FIG. 3, the primers and probes of the invention can effectively amplify ToBRFV, 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 a single 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 ToBRFV RNA at different concentrations: the total RNA of tomato leaf infected with ToBRFV obtained by extraction is detected by an ultra-micro spectrophotometer, the concentration is 92.4 ng/. Mu.L, the RNA solution is subjected to 10-fold gradient dilution, and the concentration is 92.4 and 9.24 multiplied by 10 0 、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 -8 The ng/mu L RNA solution is respectively used as templates for RT-RAA-LFA detection and common RT-PCR detection by using RNA solutions with different concentrations, and the sensitivity of the 2 methods is compared.
The common RT-PCR detection method comprises the following steps: the primer ToBRFV-F-5506/ToBRFV-R-6186 is used for carrying out a one-step RT-PCR reaction (TAKARA, beijing) on the extracted total RNA of tomato leaves 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 each of the total RNA 1.5. Mu.L, RNase Free ddH 2 O was made 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 of the conventional RT-PCR method for detecting ToBRFV RNA was 9.24X10 -4 ng/. Mu.L (FIG. 5); when ToBRFV RNA is detected by RT-RAA-LFA method, the RNA concentration is 9.24X10 -4 The colloidal gold test strip line was gradually decreased at ng/. Mu.L, but at a concentration of 9.24X10 -6 When ng/. Mu.L, the detection line can still be generated by using a colloidal gold test strip, which shows that the sensitivity of the detection by the RT-RAA-LFA method is 9.24X10 -6 ng/. Mu.L (FIG. 6), the sensitivity is improved by about 10 times compared with 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 (10)
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 as SEQ ID NO. 3.
2. The RT-RAA primer and probe combination according to claim 1, wherein the 5' end of primer ToB-RA6 sequence is labeled 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.
3. Use of the RT-RAA primer and probe combination according to claim 1 or 2 for detection of tomato brown wrinkle fruit virus.
4. Use of the RT-RAA primer and probe combination according to claim 1 or 2 for the preparation of a product for detecting tomato brown wrinkle fruit virus.
5. A kit for detecting tomato brown wrinkle virus, comprising the RT-RAA primer and probe combination according to claim 1 or 2.
6. The kit according to claim 5, wherein the kit further comprises reagents required for the RT-RAA reaction.
7. The kit of claim 6, further comprising a lateral flow chromatographic test strip.
8. 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 as set forth in claim 1 or 2;
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.
9. 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 as set forth in claim 1 or 2;
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.
10. The method according to claim 8 or 9, wherein the concentration of the primer in the reaction system used in the RT-RAA reaction is 0.12 to 0.4 μmol/L; the concentration of the probe ranges from 0.06 to 0.2 mu mol/L.
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