CN109880934B - Quarantine identification method for pepper vein mottle virus - Google Patents
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Abstract
The invention discloses a quarantine identification method of pepper vein mottle virus, which comprises the following steps: (A) Sampling and taking a sample according to the method given by SN/T2122 to obtain a sample; (B) Carrying out double-antibody sandwich enzyme-linked immunosorbent assay and RT-PCR detection on the sample; (C) And when the sample is detected, judging the result according to the following principle: performing primary screening by double-antibody sandwich enzyme-linked immunosorbent assay, and if the detection result is negative and the RT-PCR detection result is negative, determining that the sample does not carry the pepper vein mottle virus; and (3) carrying out primary screening by double-antibody sandwich enzyme-linked immunosorbent assay, and if the detection result is positive and the RT-PCR detection result is positive, judging that the sample carries the pepper vein mottle virus. The invention has good specificity, sensitivity, repeatability and reproducibility, and is not easy to generate false positive.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a quarantine and identification method for pepper vein mottle virus.
Background
Members of Potyvirus (Potyvirus) of Potyvirus family (Potyvirus) of Chilli veillan virus (ChiVMV) genus are spread by aphid in a non-persistent manner, and can be spread by means of plant sap, mechanical friction, and the like. The virus is mainly reported to infect pepper in African countries to cause serious diseases, and then, the occurrence of the virus is also reported successively in a plurality of Asian countries and regions such as African sweat, india, korea, taiwan in China and the like. In 2005, the virus was detected in Beijing in China, and then pepper vein mottle virus was also found in Hunan, shaanxi, hainan, guangxi, guangdong and other provinces. The symptoms of diseased plants in the field are mainly manifested as leaf vein distortion, chlorosis between veins to form green vein bands, mottle of leaves generally occurs, leaves become small and malformed in severe cases, fall off in advance, diseased fruits chlorosis, spot and malformation, and the like, and the yield and quality of the peppers are seriously affected.
Since Cucumber Mosaic Virus (CMV) has been reported to infect pepper, at least 45 viruses have been reported to infect pepper so far. Potyvirus (Potyvirus) is a main virus infecting pepper crops, has gradually become one of pepper head diseases in recent years, and seriously harms the development of pepper industry. The genus has nearly 200 definitive species or tentative species, and the known viruses capable of infecting Pepper include mainly 6 types of Pepper vein mottle virus (ChiVMV), pepper ringspot virus (ChiRSV), potato Virus Y (PVY), tobacco Etch Virus (TEV), pepper mottle virus (Pepper mole virus, pepMoV), and dried Pepper Vein Mottle Virus (PVMV). The existing plant virus detection method mainly comprises the technologies of PCR/RT-PCR, ELISA, electron microscope observation, gene chip and the like. However, the single use of these detection methods is often not good enough in sensitivity, specificity, repeatability and reproducibility, and false positives are likely to occur, and the pepper vein mottle virus is not detected by ELISA, so that there is a need for further improvement.
Disclosure of Invention
The invention aims to provide a pepper vein mottle virus quarantine identification method which has good specificity, sensitivity, repeatability and reproducibility and is not easy to have false positive aiming at the defects in the prior art.
The invention solves the technical problem and adopts the technical scheme that a quarantine identification method of pepper vein mottle virus is provided, which comprises the following steps:
(A) Sampling and taking a sample according to the method given by SN/T2122 to obtain a sample;
(B) Carrying out double-antibody sandwich enzyme-linked immunosorbent assay and RT-PCR detection on the sample;
(C) And when the sample is detected, judging the result according to the following principle: performing primary screening through double-antibody sandwich enzyme-linked immunosorbent assay, and if the detection result is negative and the RT-PCR detection result is negative, judging that the sample does not carry pepper mottle virus; and (3) carrying out primary screening by double-antibody sandwich enzyme-linked immunosorbent assay, and judging that the sample carries the pepper vein mottle virus if the detection result is positive and the RT-PCR detection result is positive.
Preferably, the double antibody sandwich enzyme-linked immunosorbent assay comprises the following steps:
a sample to be tested: weighing 0.5g-1.0g of sample tissue sample to be tested according to the weight ratio of 1:10 adding extraction buffer solution, grinding with mortar
Grinding into slurry, centrifuging at 4 deg.C for 10min at 10000g, and collecting supernatant as detection sample;
positive control sample: pure virus or tissue extract of pepper vein mottle virus is provided by the market and prepared by extraction buffer solution;
negative control samples: prepared by extraction buffer solution provided by the market;
blank control: using extraction buffer solution as blank control;
b) Diluting the antibody with a coating buffer solution according to the instruction, adding the diluted antibody into the holes of the enzyme-linked plate, covering the antibody with a cover, incubating at 37 ℃ for 2 h or incubating overnight in a refrigerator at 4 ℃, pouring the solution in the holes of the enzyme-linked plate, and washing with PBST for 4-6 times according to the amount of 200 mu L per hole, wherein each time lasts for 1min;
c) Respectively adding the prepared detection sample, negative control sample, positive control sample and blank control into an enzyme-linked plate, repeating at least one of the steps, covering each hole by 100 mu L, incubating at 37 ℃ for 2 h or incubating overnight in a refrigerator at 4 ℃, pouring out the solution in the holes of the enzyme-linked plate, and washing with PBST for 4-6 times according to the amount of 200 mu L per hole, wherein each time is 1min;
d) Diluting the enzyme-labeled antibody to a working concentration by using an enzyme-labeled antibody dilution buffer solution according to instructions, adding the enzyme-labeled antibody into an enzyme-linked plate, covering the enzyme-linked plate at a concentration of 100 mu L/hole, incubating the enzyme-linked plate at 37 ℃ for 2 hours, pouring out a solution in a hole of the enzyme-linked plate, and washing the enzyme-linked plate by using PBST for 4-6 times;
e) Adding a substrate PNPP into a substrate buffer solution to enable the final concentration to be 1 mg/mL, adding the substrate PNPP into an enzyme-linked plate according to 100 mu L/hole, and incubating at room temperature in a dark place;
f) Placing the enzyme linked plate in an enzyme linked instrument, and reading the OD value at 405 nm;
g) And judging the result: after the quality requirements that the OD405 value of the negative control hole is less than 0.15, the OD405 value of the positive control hole/the OD405 value of the negative control hole is more than 5-10 and the repeatability of the holes is basically consistent are met, the OD405 value of the sample/the OD405 value of the negative control hole is more than 2 obviously, and the sample is judged to be positive; if the OD405 value of the sample/the OD405 value of the negative control is near the threshold value, the sample is judged to be a suspicious sample and needs to be done again or verified by other methods; the OD405 value of the sample/the OD405 value of the negative control is obviously less than 2, and the sample is judged to be negative. Preferably, the coating antibody is prepared by diluting the pepper vein mottle virus antibody to a required concentration through a coating buffer solution.
Preferably, the enzyme-labeled antibody dilution buffer is prepared by dissolving 2.0g bovine serum albumin and 20.0g polyvinylpyrrolidone in 900 mL of 1 XPBST, diluting to 1000 mL with 1 XPBST, and storing at 4 ℃.
Preferably, the RT-PCR detection comprises the following steps:
a) Designing 2 pairs of primers ChiVMV-CP1/ChiVMV-CP2 and ChiVMV-CI3/ChiVMV-CI4 for specific amplification according to the reported ChiVMV genome sequence, wherein the base sequences of the two pairs of primers are respectively as follows:
ChiVMV-CP1：ACACCTTCTTGATTATGCTCC
ChiVMV-CP2：ATAAGGCTTCTCAGAATTGCG
ChiVMV-CI3：GGAGCCACAGAAATCAAGACA
ChiVMV-CI4：TGCCACAACTTTTCATACAAC；
wherein the target band size of the ChiVMV-CP1/ChiVMV-CP2 primer pair is 337bp, and the target band size of the ChiVMV-CI3/ChiVMV-CI4 primer pair is 662 bp;
b) And extracting total RNA: sample 200. Mu.L of the extract was placed in a 1.5 mL centrifuge tube, and 1mL Trizol test was added
Shaking for 3 min, centrifuging at 4 deg.C at 12000 g for 10min, and collecting supernatant; adding 300 μ L chloroform into the supernatant, shaking vigorously for 15 s, standing at room temperature for 5 min, centrifuging at 12000 g at 4 deg.C for 15 min, and collecting the upper water phase; adding isopropanol with the same volume into the upper water phase, reversing, mixing uniformly, standing at room temperature for 15 min, centrifuging at 12000 g at 4 deg.C for 10 min; discarding the supernatant, washing the precipitate with 1mL of 75% ethanol for 2 times, centrifuging at 4 deg.C for 7500 g for 3 min each time, and discarding the supernatant; obtaining RNA precipitate, drying, dissolving with 20-40 microliter ddH2O treated by DEPC, and storing at-20 ℃ for later use;
c) And cDNA synthesis: add 3. Mu.L of total RNA, 10. Mu. Mol/L of random primer 1. Mu.L, ddH2O to PCR tube
mu.L of the mixture is put in a water bath at 70 ℃ for 10min, quickly cooled in the ice bath for 5 min, and then the following reagents are added: 5 microliter of 5 xRT buffer solution, 2 microliter of 10 mmol/L dNTPs, 1 microliter of 200U/microliter M-MLVRT, 1 microliter of 40U/microliter RNase, 60 and 10 minutes in 42 and 70 ℃ water baths, naturally cooling to room temperature, and storing at-20 ℃ for later use;
d) And PCR amplification: during detection, the plasmid or virus-containing material containing the virus target fragment is used as a positive control, and the virus-containing material does not contain the virus target fragment
Healthy plant tissues of the virus are used as negative control, and water is used for replacing a template to be used as blank control; the PCR reaction system comprises cDNA2.0 mu L, chiVMV-CP 1.25 mu L of 10 mu mol/L, chiVMV-CP 2.25 mu L of 10 mu mol/L, chiVMV-CI 3.75 mu L of 10 mu mol/L, chiVMV-CI 4.75 mu L of 10 mu mol/L, 2 xTaq PCR mix12.5 mu L and ddH2O6.5 mu L,2 repeats are set for each reaction, and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min, then denaturation at 94 ℃ for 30 s, annealing at 53 ℃ for 45 s, extension at 72 ℃ for 1min, 35 cycles, and extension at 72 ℃ for 10min after the last cycle is finished;
e) Agarose gel electrophoresis: preparing 1.5% agarose gel, performing electrophoresis on the PCR product at voltage of 3-5V/cm,
buffer 0.5 × TBE. After electrophoresis is finished, staining the gel for 10min in 0.5 g/mL ethidium bromide solution, observing whether an expected specific DNA electrophoresis band is amplified in a gel imaging system, and photographing and recording;
f) And judging the result: if the negative control and the blank control have no specific amplification, the positive control simultaneously amplifies target bands with the size of about 337bp and 662 bp, the sample to be detected amplifies any target band (337 bp or 662 bp) consistent with the positive control or simultaneously amplifies two target bands (337 bp and 662 bp) consistent with the positive control, and then the sample is judged to be positive; if the negative control and the blank control have no specific amplification, the positive control simultaneously amplifies target bands with the sizes of about 337bp and 662 bp, and the sample to be detected does not amplify any target band (337 bp or 662 bp) consistent with the positive control, the negative is judged.
By implementing the technical scheme, the invention has the following advantages: through double-antibody sandwich enzyme-linked immunosorbent assay and RT-PCR detection, wherein during the RT-PCR detection, two pairs of specific primers are designed to carry out PCR amplification to observe whether an expected target strip is amplified, and the detection result has good specificity, sensitivity, repeatability and reproducibility and is not easy to generate false positive.
Drawings
FIG. 1 is a diagram showing the results of the specific assay according to the present invention;
FIG. 2 is a graph showing the results of the sensitivity detection according to the present invention.
Detailed Description
The following are specific embodiments of the present invention and are further described with reference to the drawings, but the present invention is not limited to these embodiments.
A quarantine and identification method of pepper vein mottle virus comprises the following steps: the method comprises the following steps:
(A) Sampling and taking a sample according to the method given by SN/T2122 to obtain a sample;
(B) And carrying out double-antibody sandwich enzyme-linked immunosorbent assay and RT-PCR detection on the sample.
1. The double-antibody sandwich enzyme-linked immunosorbent assay method comprises the following steps:
a) And a sample to be detected: weighing 0.5g-1.0g of sample tissue sample to be tested according to the weight ratio of 1:10 (weight: volume, W/V) adding extraction buffer solution, grinding into slurry by using a mortar, centrifuging for 10min at 10000g at 4 ℃, and taking supernatant as a detection sample;
positive control sample: pure virus or tissue extract of pepper vein mottle virus is provided by the market and prepared by extraction buffer solution;
negative control samples: the pepper leaves which are not infected with pepper vein mottle virus are provided by the market and prepared by using an extraction buffer solution;
blank control: using extraction buffer solution as blank control;
b) Diluting the antibody with a coating buffer solution according to the specification, adding the diluted antibody into the holes of the enzyme-linked plate, covering the antibody per hole, incubating at 37 ℃ for 2 h or incubating overnight in a refrigerator at 4 ℃, pouring out the solution in the holes of the enzyme-linked plate, and washing with PBST for 4-6 times according to the amount of 200 mu L per hole, wherein each time lasts for 1min;
c) Respectively adding the prepared detection sample, negative control sample, positive control sample and blank control into an enzyme-linked plate, repeating at least one of the steps, covering each hole by 100 mu L, incubating at 37 ℃ for 2 h or incubating overnight in a refrigerator at 4 ℃, pouring out the solution in the holes of the enzyme-linked plate, and washing with PBST for 4-6 times according to the amount of 200 mu L per hole, wherein each time is 1min;
d) Diluting the enzyme-labeled antibody to a working concentration by using an enzyme-labeled antibody dilution buffer solution according to a specification, adding the enzyme-labeled antibody into an enzyme-linked plate, covering the enzyme-linked plate, incubating the enzyme-linked plate at 37 ℃ for 2 hours, pouring out a solution in a hole of the enzyme-linked plate, and washing the enzyme-linked plate by PBST for 4 to 6 times;
e) Adding a substrate PNPP into a substrate buffer solution to enable the final concentration to be 1 mg/mL, adding the substrate PNPP into an enzyme-linked plate according to 100 mu L/hole, and incubating at room temperature in a dark place;
f) Placing the enzyme linked plate in an enzyme linked instrument, and reading the OD value at 405 nm;
g) And judging the result: after the quality requirements that the OD405 value of the negative control hole is less than 0.15, the OD405 value of the positive control hole/the OD405 value of the negative control hole is more than 5-10 and the repeatability of the holes is basically consistent are met, the OD405 value of the sample/the OD405 value of the negative control hole is more than 2 obviously, and the sample is judged to be positive; the sample OD405 value/negative control OD405 value is near the threshold value, and the sample is judged to be a suspicious sample and needs to be done again or verified by other methods; the OD405 value of the sample/the OD405 value of the negative control are obviously less than 2, and the sample is judged to be negative.
Wherein, the 1 XPBST buffer (pH 7.4) is prepared by dissolving 8.0 g of sodium chloride, 0.2 g of potassium dihydrogen phosphate, 1.15 g of disodium hydrogen phosphate, 0.2 g of potassium chloride and 0.5 mL of Tween-20 in 900 mL of sterile double distilled water and diluting to 1mL of water to 4 ℃ for storage.
The extraction buffer (pH 7.4) was prepared by dissolving 1.3 g of sodium sulfite and 20.0g of polyvinylpyrrolidone in 900 mL of 1 XPBST and diluting to 1mL with 1 XPBST and storing at 4 ℃.
The enzyme-labeled antibody dilution buffer was prepared by dissolving 2.0g bovine serum albumin and 20.0g polyvinylpyrrolidone in 900 mL of 1 XPBST, diluting to 1000 mL with 1 XPBST, and storing at 4 ℃.
The substrate buffer (pH 9.8) was prepared by dissolving 97 mL diethanolamine and 0.1 g magnesium chloride in 800 mL sterile double distilled water, adjusting the pH to 9.8 with concentrated HCl, diluting to 1000 mL, and storing at 4 ℃.
Through the method, sampling and sampling are carried out according to the method given by SN/T2122, 10 leaves of different hot peppers are obtained and used as samples to carry out double-antibody sandwich enzyme-linked immunosorbent assay, and the results are shown in the following table 1:
2. RT-PCR detection steps
A) Designing 2 pairs of primers ChiVMV-CP1/ChiVMV-CP2 and ChiVMV-CI3/ChiVMV-CI4 for specific amplification according to the reported ChiVMV genome sequence (CP and CI gene), wherein the base sequences of the two pairs of primers are respectively as follows:
ChiVMV-CP1：ACACCTTCTTGATTATGCTCC
ChiVMV-CP2：ATAAGGCTTCTCAGAATTGCG
ChiVMV-CI3：GGAGCCACAGAAATCAAGACA
ChiVMV-CI4：TGCCACAACTTTTCATACAAC。
wherein, the target band size of the ChiVMV-CP1/ChiVMV-CP2 primer pair is 337bp, and the target band size of the ChiVMV-CI3/ChiVMV-CI4 primer pair is 662 bp.
B) And extracting total RNA: putting 200 mu L of sample extracting solution into a 1.5 mL centrifuge tube, adding 1mL Trizol reagent, shaking up for 3 min with intense shaking, centrifuging at 12000 g at 4 ℃ for 10min, and taking supernatant; adding 300 μ L chloroform into the supernatant, shaking for 15 s, standing at room temperature for 5 min, centrifuging at 12000 g at 4 deg.C for 15 min, and collecting the upper water phase; adding isopropanol with the same volume into the upper water phase, reversing, mixing uniformly, standing at room temperature for 15 min, centrifuging at 12000 g at 4 deg.C for 10 min; discarding the supernatant, washing the precipitate with 1mL of 75% ethanol for 2 times, centrifuging at 4 deg.C for 7500 g for 3 min each time, and discarding the supernatant; obtaining RNA precipitate, drying, dissolving with 20-40 mu L of DDH2O treated by DEPC, and storing at-20 ℃ for later use;
c) And cDNA synthesis: add 3. Mu.L total RNA, 10. Mu. Mol/L random primer 1. Mu.L, ddH2O 7. Mu.L to PCR tube, water bath at 70 ℃ for 10min, ice bath for 5 min rapidly, add the following reagents: 5 microliter of 5 xRT buffer solution, 2 microliter of 10 mmol/L dNTPs, 1 microliter of 200U/microliter M-MLVRT, 1 microliter of 40U/microliter RNase, 60 and 10 minutes in 42 and 70 ℃ water baths, naturally cooling to room temperature, and storing at-20 ℃ for later use;
d) And PCR amplification: during detection, plasmid or virus-containing material containing a virus target fragment is used as a positive control, healthy plant tissues without virus are used as a negative control, and water is used as a blank control instead of a template; the PCR reaction system comprises cDNA2.0. Mu.L, chiVMV-CP 1.25. Mu.L at 10. Mu.mol/L, chiVMV-CP 2.25. Mu.L at 10. Mu.mol/L, chiVMV-CI 3.75. Mu.L at 10. Mu.mol/L, chiVMV-CI 4.75. Mu.L at 10. Mu.mol/L, 2 XTaq PCR mix 12.5. Mu.L and ddH2O6.5. Mu.L, and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min, then denaturation at 94 ℃ for 30 s, annealing at 53 ℃ for 45 s, extension at 72 ℃ for 1min, 35 cycles, and extension at 72 ℃ for 10min after the last cycle is finished;
e) Agarose gel electrophoresis: a1.5% agarose gel was prepared and the PCR products were electrophoresed at a voltage of 3-5V/cm in a buffer of 0.5 XTBE. After electrophoresis is finished, staining the gel for 10min in 0.5 g/mL ethidium bromide solution, observing whether an expected specific DNA electrophoresis band is amplified in a gel imaging system, and photographing and recording;
f) And judging the result: if the negative control and the blank control have no specific amplification, the positive control simultaneously amplifies target bands with the sizes of about 337bp and 662 bp, the sample to be detected amplifies any target band (337 bp or 662 bp) consistent with the positive control or simultaneously amplifies two target bands (337 bp and 662 bp) consistent with the positive control, the positive control is judged; if the negative control and the blank control have no specific amplification, the positive control simultaneously amplifies target bands with the sizes of about 337bp and 662 bp, and the sample to be detected does not amplify any target band (337 bp or 662 bp) consistent with the positive control, the sample is judged to be negative.
Wherein the 0.5 XTBE buffer is prepared by mixing 54.0 g Tris base, 27.5 g boric acid and 20mL of 0.5 mol/L EDTA (pH 8.0), adding distilled water to 1L, and diluting to 0.5 XTBE with distilled water.
The leaf of 10 parts of pepper in1 was used as a sample for RT-PCR detection, and the detection results are shown in the following table 2:
(C) The result judgment principle of the double-antibody sandwich enzyme-linked immunosorbent assay and RT-PCR detection method is as follows:
1) And performing primary screening by double-antibody sandwich enzyme-linked immunosorbent assay, and if the detection result is negative and the RT-PCR detection result is negative, judging that the sample does not carry ChiVMV.
2) And performing primary screening by double-antibody sandwich enzyme-linked immunosorbent assay, and if the detection result is positive and the RT-PCR detection result is positive, determining that the sample carries ChiVMV.
Whether the 10 samples carried ChiVMV by the above test is shown in Table 3 below:
(D) And recording various experimental data including sample sources, types, time, experimental time, places, methods, results and the like, and signing by hands and experimenters. The serological detection result retains a light absorption value data report, and the molecular biological detection result retains an electrophoresis picture.
(E) And the sample judged to be positive by the detection result is properly stored in an ultra-low temperature refrigerator at the temperature of between 20 ℃ below zero and 80 ℃ below zero, and is registered and marked for rechecking.
In addition, a specificity test and a sensitivity test for RT-PCR detection are performed
(1) And materials: cDNA (ChiVMV, A-E are cucumber mosaic virus (CMW), tobacco Mosaic Virus (TMV), potato Virus Y (PVY), pepper mottle virus (PMMoV) respectively), primer ChiVMV-CP1/ChiVMV-CP2 (ChiVMV-CP 1: ACACCTTCTTGATGCTCC, chiVMV-CP2: ATAAGGCTTCTCAGAATTGCG)
(2) Method of producing a semiconductor device
1) The PCR reaction system is shown in Table 4, and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min, then denaturation at 94 ℃ for 30 s, annealing at 53 ℃ for 45 s, extension at 72 ℃ for 1min, 35 cycles, and extension at 72 ℃ for 10min after the last cycle.
TABLE 4
2) Agarose gel electrophoresis: a1.5% agarose gel was prepared and the PCR products were electrophoresed at a voltage of 3-5V/cm in a buffer of 0.5 XTBE. After electrophoresis is finished, staining the gel for 10min by 0.5 g/mL ethidium bromide solution, observing whether an expected specific DNA electrophoresis band is amplified in a gel imaging system, photographing and recording.
3) And the results are judged
If the sample PCR amplified the target fragment with the size of about 337bp, the detection result is judged to be positive.
(3) Specificity test
The procedure of (2) was carried out using cDNAs of ChiVMV and A-E as templates.
As shown in FIG. 1, the specific detection results show that the method can not amplify expected bands from cucumber mosaic virus (CMW), tobacco Mosaic Virus (TMV), potato Virus Y (PVY) and pepper mild mottle virus (PMMoV), can amplify expected 337bp bands from ChiVMV, and shows good specificity.
4) And sensitivity test
Taking cDNA stock solution to perform 10-fold gradient dilution to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 The procedure of (2) was followed using the stock solution and each dilution as templates.
As shown in FIG. 2, the detection of ChiVMV cDNA in serial dilution by this method showed that dilution 10 could be clearly detected by this method -2 The cDNA template has good sensitivity.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments, or alternatives may be employed, by those skilled in the art, without departing from the spirit or ambit of the invention as defined in the appended claims.
Sequence listing
<110> Fuqing entry-exit inspection and quarantine bureau integrated technology service center
<120> quarantine identification method for pepper vein mottle virus
<160> 4
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial sequence
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<223> ChiVMV-CP1
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acaccttctt gattatgctc c 21
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<212> DNA
<213> Artificial sequence
<220>
<222> (1)..(21)
<223> ChiVMV-CP2
<400> 2
ataaggcttc tcagaattgc g 21
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<222> (1)..(21)
<223> ChiVMV-CI3
<400> 3
ggagccacag aaatcaagac a 21
<210> 4
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<212> DNA
<213> Artificial sequence
<220>
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<223> ChiVMV-CI4
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tgccacaact tttcatacaa c 21
Claims (1)
1. A quarantine identification method of pepper vein mottle virus is characterized by comprising the following steps:
(A) Sampling and taking a sample according to the method given by SN/T2122 to obtain a sample;
(B) Carrying out double-antibody sandwich enzyme-linked immunosorbent assay and RT-PCR detection on the sample;
(C) And when the sample is detected, judging the result according to the following principle: performing primary screening by double-antibody sandwich enzyme-linked immunosorbent assay, and if the detection result is negative and the RT-PCR detection result is negative, determining that the sample does not carry the pepper vein mottle virus; performing primary screening by double-antibody sandwich enzyme-linked immunosorbent assay, and if the detection result is positive and the RT-PCR detection result is positive, determining that the sample carries the pepper vein mottle virus;
the double-antibody sandwich enzyme-linked immunosorbent assay comprises the following steps:
a) And a sample to be detected: weighing 0.5g-1.0g of sample tissue to be tested according to the weight ratio of 1:10 adding an extraction buffer solution, grinding into slurry by using a mortar, centrifuging for 10min at the temperature of 4 ℃ by 10000g, and taking supernate, namely a detection sample;
positive control sample: pure virus or tissue extract of pepper vein mottle virus is provided by the market and prepared by using an extraction buffer solution;
negative control samples: is prepared by extraction buffer solution provided by the market;
blank control: using extraction buffer solution as blank control;
b) Diluting the antibody with a coating buffer solution according to the instruction, adding the diluted antibody into the holes of the enzyme-linked plate, covering the antibody with a cover, incubating at 37 ℃ for 2 h or incubating overnight in a refrigerator at 4 ℃, pouring the solution in the holes of the enzyme-linked plate, and washing with PBST for 4-6 times according to the amount of 200 mu L per hole, wherein each time lasts for 1min;
c) Respectively adding the prepared detection sample, negative control sample, positive control sample and blank control into an enzyme-linked plate, repeating at least one of the steps, covering each hole by 100 mu L, incubating at 37 ℃ for 2 h or incubating overnight in a refrigerator at 4 ℃, pouring out the solution in the holes of the enzyme-linked plate, and washing with PBST for 4-6 times according to the amount of 200 mu L per hole, wherein each time is 1min;
d) Diluting the enzyme-labeled antibody to a working concentration by using an enzyme-labeled antibody dilution buffer solution according to instructions, adding the enzyme-labeled antibody into an enzyme-linked plate, covering the enzyme-linked plate at a concentration of 100 mu L/hole, incubating the enzyme-linked plate at 37 ℃ for 2 hours, pouring out a solution in a hole of the enzyme-linked plate, and washing the enzyme-linked plate by using PBST for 4-6 times;
e) Adding a substrate PNPP into a substrate buffer solution to enable the final concentration to be 1 mg/mL, adding the substrate PNPP into an enzyme-linked plate according to 100 mu L/hole, and incubating at room temperature in a dark place;
f) Placing the enzyme-linked plate in an enzyme-linked instrument, and reading the OD value at 405 nm;
g) And judging the result: after the quality requirements that the OD405 value of the negative control hole is less than 0.15, the OD405 value of the positive control hole/the OD405 value of the negative control hole is more than 5-10 and the repeatability of the holes is basically consistent are met, the OD405 value of the sample/the OD405 value of the negative control hole is obviously more than 2, and the sample is judged to be positive; if the OD405 value of the sample/the OD405 value of the negative control is near the threshold value, the sample is judged to be a suspicious sample and needs to be done again or verified by other methods; the OD405 value of the sample/the negative control OD405 value is obviously less than 2, and the sample is judged to be negative;
the preparation method of the enzyme-labeled antibody dilution buffer solution comprises the steps of dissolving 2.0g of bovine serum albumin and 20.0g of polyvinylpyrrolidone in 900 mL of 1 xPBST, fixing the volume to 1000 mL by using 1 xPBST, and storing at 4 ℃;
the RT-PCR detection comprises the following steps:
a) Designing 2 pairs of primers ChiVMV-CP1/ChiVMV-CP2 and ChiVMV-CI3/ChiVMV-CI4 for specific amplification according to the reported ChiVMV genome sequence, wherein the base sequences of the two pairs of primers are respectively as follows:
ChiVMV-CP1：ACACCTTCTTGATTATGCTCC
ChiVMV-CP2：ATAAGGCTTCTCAGAATTGCG
ChiVMV-CI3：GGAGCCACAGAAATCAAGACA
ChiVMV-CI4：TGCCACAACTTTTCATACAAC；
wherein, the target band size of the ChiVMV-CP1/ChiVMV-CP2 primer pair is 337bp, and the target band size of the ChiVMV-CI3/ChiVMV-CI4 primer pair is 662 bp;
b) And extracting total RNA: putting 200 μ L of sample extract into a 1.5 mL centrifuge tube, adding 1mL Trizol reagent, shaking for 3 min, centrifuging at 4 deg.C at 12000 g for 10min, and collecting supernatant; adding 300 μ L chloroform into the supernatant, shaking for 15 s, standing at room temperature for 5 min, centrifuging at 12000 g at 4 deg.C for 15 min, and collecting the upper water phase; adding isopropanol with the same volume into the upper water phase, reversing, mixing uniformly, standing at room temperature for 15 min, centrifuging at 12000 g at 4 deg.C for 10 min; discarding the supernatant, washing the precipitate with 1mL of 75% ethanol for 2 times, centrifuging at 4 deg.C for 7500 g for 3 min each time, and discarding the supernatant; obtaining RNA precipitate, drying, dissolving with 20-40 mu L of DDH2O treated by DEPC, and storing at-20 ℃ for later use;
c) And cDNA synthesis: add 3. Mu.L total RNA, 10. Mu. Mol/L random primer 1. Mu.L, ddH2O 7. Mu.L to PCR tube, water bath at 70 ℃ for 10min, ice bath for 5 min rapidly, add the following reagents: 5 microliter of 5 xRT buffer solution, 2 microliter of 10 mmol/L dNTPs, 1 microliter of 200U/. Mu.L M-MLVRT, 1 microliter of 40U/. Mu.L RNase, 60 microliter of 42 ℃ water bath, 10 microliter of 70 ℃ water bath, naturally cooling to room temperature, and storing at-20 ℃ for later use;
d) And PCR amplification: during detection, a plasmid containing a virus target fragment or a virus-containing material is used as a positive control, a healthy plant tissue without virus is used as a negative control, and water is used as a blank control to replace a template; the PCR reaction system comprises cDNA2.0 mu L, chiVMV-CP 1.25 mu L of 10 mu mol/L, chiVMV-CP 2.25 mu L of 10 mu mol/L, chiVMV-CI 3.75 mu L of 10 mu mol/L, chiVMV-CI 4.75 mu L of 10 mu mol/L, 2 xTaq PCR mix12.5 mu L and ddH2O6.5 mu L,2 repeats are set for each reaction, and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min, then denaturation at 94 ℃ for 30 s, annealing at 53 ℃ for 45 s, extension at 72 ℃ for 1min, 35 cycles, and extension at 72 ℃ for 10min after the last cycle is finished;
e) Agarose gel electrophoresis: preparing 1.5% agarose gel, carrying out electrophoresis on the PCR product, carrying out electrophoresis at the voltage of 3-5V/cm and the buffer solution of 0.5 xTBE, after the electrophoresis is finished, staining the PCR product for 10min in 0.5 g/mL ethidium bromide solution, observing whether an expected specific DNA electrophoresis band is amplified or not in a gel imaging system, and taking a picture and recording;
f) And judging the result: if the negative control and the blank control have no specific amplification, the positive control simultaneously amplifies target bands with the sizes of 337bp and 662 bp, the sample to be detected amplifies any one of the target bands consistent with the positive control or two of the target bands consistent with the positive control simultaneously, and then the sample is judged to be positive; and if the negative control and the blank control have no specific amplification, the positive control simultaneously amplifies target bands with the sizes of 337bp and 662 bp, and the sample to be detected does not amplify any target band consistent with the positive control, the sample is judged to be negative.
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