Double-antibody sandwich enzyme-linked immunosorbent assay kit for grape berry necrosis virus, preparation method and detection method

Abstract

The invention belongs to the field of biotechnology products for plant virus detection, and particularly relates to a double-antibody sandwich enzyme-linked immunosorbent assay kit for grape berry necrosis viruses, a preparation method and a detection method.The double-antibody sandwich enzyme-linked immunosorbent assay kit comprises a microwell plate for ELISA, an enzyme-labeled antibody, a buffer solution, a developing solution and a stop buffer, the microwell plate is coated with a GINV specific polyclonal antibody, and the microwell plate is coated with a GINV specific polyclonal antibody. The enzyme-labeled antibody is a GINV specific polyclonal antibody labeled by horse radish peroxidase. The immunogen adopted by the invention is a GINV coat protein antigen expressed by genetic engineering. The invention provides a rapid, sensitive, simple and convenient method for detecting the grape berry necrosis virus on the serological level, is economical and practical, and also provides an effective method for preventing and treating the grape berry necrosis virus in grape production and preparing virus-free seedlings of the grape berry necrosis virus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology products for plant virus detection, and specifically relates to a grape berry necrosis virus double antibody sandwich enzyme-linked immunosorbent assay kit, a preparation method and a detection method. Background Art

[0002] Grapevine berry inner necrosis virus (GINV) is an RNA virus of the genus Trichovirus in the family Betaflexiviridae. The virus particle size is 740 nm × 12 nm, and the genome is 7229 nt long. It consists of three open reading frames, encoding replicase protein (RP), move protein (MP) and coat protein (CP). GINV is considered to be the pathogen of grape berry inner necrosis (originally called mosaic disease). The disease was first discovered on Kyoho grapes in Japan in 1984 and caused serious damage to vineyards in Yamanashi Prefecture, Japan at that time. Infected plants have weakened growth, delayed spring budding, and show symptoms such as stem necrosis, short nodes, chlorosis of leaves, smaller berries, discoloration of the outside of the fruit, and necrosis inside the fruit. It is reported that the virus is common in my country's grape-producing areas. Grape plants infected with GINV grow slowly, show internal necrosis on branches, shortened internodes, mosaic leaves, and grape berries show external discoloration and internal necrosis. GINV can be spread in the field through gall mites. At present, GINV is mainly detected by electron microscopy and molecular biology techniques. There is no economical and rapid detection method. In order to reduce the harm of GINV to my country's grape industry, it is necessary to establish a sensitive and efficient detection system. Since there is no GINV-specific antiserum, it cannot be detected by enzyme-linked immunosorbent assay (ELISA). Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides a grape berry necrosis virus double antibody sandwich enzyme-linked immunosorbent assay kit, a preparation method and a detection method. The detection using the kit is accurate and efficient, easy to promote, economical and practical.

[0004] The technical solution provided by the present invention is as follows:

[0005] A double antibody sandwich enzyme-linked immunosorbent assay kit for grape berry necrosis virus, the kit comprising a GINV-specific polyclonal antibody, the GINV-specific polyclonal antibody being coated on a polyclonal antibody-coated plate, the immunogenic amino acid sequence of the GINV-specific polyclonal antibody being: MSIRQELRDLVRRELITKFSEDKQVLHGLNENQQSLVLDHIFGNIAIEGTSEETVYPQSATQCKEGFDPTMPVIREYNLSSVVNKIRIYKESHKNEDIKSMTFRQICASFAIDAKLGLQKFNRYGVHSNIYKKHPKICDKAPEMAFDFNDGLNFNLLTKQQKTVIQNLNQLLFRVESEKQKTNAITSSSEGAFTM (SEQ ID No. 1).

[0006] Furthermore, the kit also includes horseradish peroxidase labeled polyclonal antibody, genetically engineered recombinant GINV coat protein standard, and quality control product.

[0007] Furthermore, the kit also includes auxiliary reagents: extraction reaction solution, washing buffer, enzyme buffer, color developer and reaction termination solution.

[0008] The present invention also provides a method for preparing the above-mentioned grape berry necrosis virus double antibody sandwich enzyme-linked immunosorbent assay kit, which includes the steps of preparing a genetically engineered recombinant GINV coat protein standard, preparing a GINV-specific polyclonal antibody, preparing a polyclonal antibody coated plate, preparing a horseradish peroxidase-labeled polyclonal antibody, preparing a quality control product, and preparing an auxiliary reagent.

[0009] Furthermore, in the preparation step of the GINV capsid protein standard, the total RNA of grape leaves infected with GINV was used as a template to amplify the GINV CP gene, cloned into the pET-30a vector to obtain the recombinant plasmid pET30a-GINV-CP, and the 27 kDa capsid protein was obtained by prokaryotic expression and purification.

[0010] Furthermore, the nucleotide sequences of the primers used to amplify the CP gene of GINV are as follows: GINV-CP-F: 5'-gatatcggatccgaattcatgtcaattcgtcaggaactgag-3' GINV-CP-R: 5'-ctcgagtgcggccgcaagcttttacatagtaaaagcaccctcgc-3'.

[0011] Furthermore, in the step of preparing the GINV-specific polyclonal antibody, the GINV-specific polyclonal antibody is obtained by immunizing an animal with GINV coat protein.

[0012] Furthermore, in the step of preparing the polyclonal antibody coated plate, the GINV-specific polyclonal antibody obtained by immunizing animals with purified GINV capsid protein is used to coat the ELISA plate.

[0013] Furthermore, in the step of preparing the horseradish peroxidase-labeled polyclonal antibody, the GINV-specific polyclonal antibody obtained by immunizing an animal with purified GINV coat protein is labeled with horseradish peroxidase.

[0014] Specifically, a double antibody sandwich enzyme-linked immunosorbent assay kit for detecting grape berry necrosis virus includes a 96-well microplate coated with GINV-specific polyclonal antibodies (i.e., a polyclonal antibody-coated plate), horseradish peroxidase-labeled polyclonal antibodies, genetically engineered recombinant GINV capsid protein standards, quality control products, and auxiliary reagents such as substrate reaction solution, washing buffer, enzyme buffer, color developer, and reaction termination solution.

[0015] In the step of preparing the standard product of the capsid protein of the grape berry necrosis virus of the present invention, the total RNA of grape leaves infected with GINV is used as a template to amplify the GINV CP The gene was cloned into the pET-30a vector after restriction digestion and ligation to obtain the recombinant plasmid pET30a-GINV-CP, which was transformed into the Escherichia coli BL21 strain, cultured at 37°C overnight, induced by IPTG, and then purified by nickel column affinity chromatography to obtain a capsid protein with a size of 27 kDa.

[0016] Amplification of GINV CP The nucleotide sequences of the primers used for the gene are: GINV-CP-F: 5'-gatatcggatccgaattcatgtcaattcgtcaggaactgag-3' GINV-CP-R: 5'-ctcgagtgcggccgcaagcttttacatagtaaaagcaccctcgc-3'.

[0017] In the preparation step of the polyclonal antibody coated plate of the present invention, the polyclonal antibody coated ELISA plate obtained by immunizing New Zealand white rabbits with prokaryotic expression and purification of GINV coat protein (i.e., the coat protein obtained in the preparation step of the grape berry necrosis virus coat protein standard) can be prepared. The ELISA plate used in the kit is an imported 96-well ELISA plate, and the preparation steps are as follows:

[0018] (1) Select young and middle-aged New Zealand white rabbits weighing about 2.5 kg and mark them. Use GINV coat protein at a concentration of 1 mg / mL as the immunogen. In the first immunization, the antigen is first mixed thoroughly, and then mixed thoroughly with Freund's complete adjuvant at a volume ratio of 1:1. After complete emulsification, multiple subcutaneous injections are performed, with 0.2 mL per point. Freund's incomplete adjuvant is used for the second to fourth immunizations, and it is still mixed thoroughly with the antigen at a volume ratio of 1:1. The second immunization is performed on the 14th day after the first immunization, and the interval between the second and third immunizations is 7 days. On the 7th day after the third immunization, a small sample of serum is collected from the middle ear artery of the rabbit for testing. If the test is qualified, an additional immunization is performed 7 days later. Whole blood can be collected 7 days after the additional immunization. After blood collection, sodium azide with a mass percentage concentration of 0.02% is added and stored at -20°C. The obtained antiserum is purified by DE52 anion exchange column chromatography.

[0019] (2) Dilute the purified rabbit anti-polyclonal antibody with pH 9.6, 50 mM phosphate buffer to a final concentration of 1 μg / mL, then add 100 μL to each well of the ELISA plate, and incubate at 37°C for 2 h. Rinse the ELISA plate with washing buffer and then block it with blocking solution. The blocking solution is a phosphate buffer containing 2wt% gelatin and 0.5wt% bovine serum albumin. After blocking, spin dry and air dry to obtain a polyclonal antibody coated plate.

[0020] In the preparation step of the horseradish peroxidase-labeled polyclonal antibody of the present invention, horseradish peroxidase and the polyclonal antibody obtained by immunizing New Zealand white rabbits with purified GINV coat protein are labeled according to a 1:1 molar molecular ratio. The preparation steps are as follows:

[0021] (1) Dissolve horseradish peroxidase in distilled water, add the freshly prepared NaIO4 aqueous solution, mix well, and incubate at 4°C in the dark for 30 min.

[0022] (2) Add 0.5 mL of ethylene glycol aqueous solution and incubate at 25°C in the dark for 30 min.

[0023] (3) Add an aqueous solution containing purified antibody (in an equal molar ratio to horseradish peroxidase), mix well, place in a dialysis bag, and dialyze against 0.05 M, pH 9.6 carbonate buffer for 6 h;

[0024] (4) Add NaBH4 aqueous solution, mix well, and incubate at 4°C in the dark for 2 h.

[0025] (5) Slowly add an equal volume of saturated ammonium sulfate solution to the above solution, mix well, let stand at 4°C for 30 min, centrifuge, remove the supernatant, add 20 μL of 0.02 M phosphate buffer (pH 7.4) to dissolve the precipitate, and dialyze to desalt overnight at 4°C;

[0026] (6) Centrifuge and take the supernatant, which is the enzyme-antibody conjugate. Add an equal volume of glycerol and store it at -20°C after packaging.

[0027] The auxiliary reagents of the present invention include washing buffer, enzyme buffer, extraction buffer, color developing agent and reaction termination solution. The preparation method of the auxiliary reagents is as follows:

[0028] (1) Extraction buffer: 0.87wt% potassium sulfate, 0.5wt% polyvinylpyrrolidone, 0.05wt% Tween-20, 0.1wt% mercaptoethanol, 0.58wt% sodium chloride, 0.2wt% bovine serum albumin, and Tris hydrochloric acid buffer (pH 7.4) containing 0.02wt% sodium azide;

[0029] (2) Enzyme buffer, prepared with distilled water, containing 1 wt% polyethylene glycol, 0.5 wt% bovine serum albumin, 0.01 wt% sodium thimerosal, and phosphate buffer (pH 7.4) containing 0.05 wt% Tween-20;

[0030] (3) Washing buffer: phosphate buffer containing 0.05 wt% Tween-20, pH 7.2;

[0031] (4) Color developer A, containing a final concentration of 1.46 wt % sodium dihydrogen phosphate, 0.93 wt % citric acid, and 0.045 wt % hydrogen peroxide aqueous solution;

[0032] (5) Color developer B, containing an aqueous solution with a final concentration of 0.03 wt % tetramethylbenzidine, 0.96 wt % citric acid, 0.019 wt % ethylenediaminetetraethylene glycol sodium salt, 2 wt % DMSO and 4 wt % glycerol;

[0033] (6) The stop solution is 2 M H2SO4 solution prepared in distilled water.

[0034] In the preparation step of the positive quality control product in the kit of the present invention, the grape leaves that are positive in the RT-PCR test for the grape berry necrosis virus (referring to the grape berry necrosis virus being positive) are ground with the above-mentioned extraction buffer and then centrifuged, and the supernatant is taken as the positive quality control product. In the preparation step of the negative quality control product, the grape leaves that are negative in the RT-PCR test for the grape berry necrosis virus are ground with the extraction buffer and then centrifuged, and the supernatant is taken as the negative quality control product.

[0035] After adopting the above scheme, the present invention prepares a double antibody sandwich enzyme-linked immunosorbent assay kit comprising a box body, an ELISA plate, and various reagents.

[0036] The present invention also provides a method for detecting grape berry necrosis virus, and uses the above-mentioned kit to detect the grape berry necrosis virus.

[0037] Furthermore, the detection method comprises the following steps:

[0038] (1) Grind the grape leaf sample to be tested with extraction buffer, centrifuge it, and aspirate the supernatant;

[0039] (2) Add the supernatant to the polyclonal antibody-coated plate at 100 μL / well and incubate at 37°C for 30 min:

[0040] (3) Add 1 μg / mL enzyme-labeled antibody and incubate at 37°C for 2 h;

[0041] (4) Mix equal volumes of colorimetric solutions A and B, add 100 μL / well, and incubate at 37°C for 15 min.

[0042] (5) Add 50 μL / well of stop solution and measure the OD value of each well on a microplate reader at 450 nm within 10 min. 450nm If the ratio is greater than 2.1, it is judged as positive.

[0043] The detection method of the invention can detect the coat protein of grape berry necrosis virus to diagnose whether grapes are infected with grape berry necrosis virus, and the sample to be tested is extracted from leaf tissue.

[0044] Beneficial Effects

[0045] The present invention provides a double antibody sandwich enzyme-linked immunosorbent assay kit for detecting grape berry necrosis virus, a preparation method and a detection method. The kit adopts a high-purity coat protein expressed by genetic engineering as an immunogen, has high sensitivity, good specificity, and is simple to operate. It is economical and applicable, suitable for large-scale detection in production, conducive to large-scale commercial promotion, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the electrophoresis diagram of GINV coat protein expression and purification;

[0047] Figure 2 This is a schematic diagram of the test results and significance analysis of 21 grape leaf samples in the field. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0049] (1) Prokaryotic expression and purification of GINV capsid protein

[0050] ① Total RNA extraction (polysaccharide and polyphenol plant tissue lysis method)

[0051] ② Primer design

[0052] According to the multiple GINV sequences registered on NCBI, primers were designed in the conserved regions to synthesize and amplify the specific primers for the GINV coat protein nucleotide sequence: GINV-CP-F: 5'-gatatcggatccgaattcatgtcaattcgtcaggaactgag-3' (SEQ ID No. 2) and GINV-CP-R: 5'-ctcgagtgcggccgcaagcttttacatagtaaaagcaccctcgc-3' (SEQ ID No. 3); restriction enzyme sites were introduced respectively EcoR I and XOt I, to connect the prokaryotic expression recombinant vector pET-30a. The primers were purified by PAGE and sent to Shanghai Shenggong Biotechnology Co., Ltd. for synthesis.

[0053] ③ RT-PCR

[0054] Total RNA extracted from grape leaves collected in Taizhou was used as a template, and cDNA was synthesized using GINV-CP-R primers and reverse transcriptase M-MLV from TAKARA. The reverse transcription system is shown in Table 1.

[0055] Table 1 Reverse transcription system

[0056]

[0057] Mix by flicking the tube gently, centrifuge briefly, incubate at 42°C for 60 min, and then place on ice until use;

[0058] The PCR reaction system is shown in Table 2.

[0059] Table 2 PCR reaction system

[0060]

[0061] The PCR program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, for a total of 28 cycles, extension at 72°C for 5 min, and storage at 4°C;

[0062] The RT-PCR product was aspirated for 1% agarose gel electrophoresis, and the gel was cut and recovered.

[0063] ④Construction of recombinant plasmid

[0064] Recycling product utilization EcoR I and XOt I was double-digested, and the digestion recovery product was connected to the corresponding digested pET-30a vector, transformed into Escherichia coli DH5α competent cells, and positive recombinant bacteria were identified by bacterial liquid PCR, digestion and sequencing.

[0065] The enzyme digestion system is shown in Table 3.

[0066] Table 3 Enzyme digestion system

[0067]

[0068] ⑤ Prokaryotic expression of protein

[0069] By AxyPrep TM The positive recombinant plasmid was extracted with DNA Gel Extraction kit and transformed into competent cells of Escherichia coli BL21 strain. Positive clones were obtained by bacterial liquid PCR and enzyme digestion identification.

[0070] Select the positive strains and inoculate them into 2 mL LB liquid medium and activate them at 37°C overnight. Add the activated bacterial solution into fresh LB at a ratio of 1:100 and culture at 37°C until OD 600 0.6. Induce at 28℃ and 37℃, take 2 tubes at each temperature, add IPTG to a final concentration of 1 mM in one tube, and do not add IPTG in the other tube as a control, and culture for 4-6 h. Take 2 mL of the induced culture and centrifuge at 12 000rpm for 30 s to collect the bacteria. Add 200 μL SDS-PAGE Loading Buffer, boil in boiling water for 10 min, and centrifuge at 12 000rpm for 10 min. Take 8 mL of the supernatant for SDS-PAGE electrophoresis.

[0071] After confirming protein expression through preliminary experiments, add 10 mL of activated bacterial solution to 1 L of liquid LB at a ratio of 1:100 and culture at 37°C until OD 600 The p-value was 0.6, and 1 mM IPTG was used to induce at 18°C ​​for 18 h. Then the bacteria were collected by centrifugation, resuspended in 40 mL of protein buffer containing 20 mM Tri-HCl (pH 6.8), 500 mM NaCl, and 10% glycerol, and protease inhibitors were added to a final concentration of 1 mM. Ultrasonic disruption was performed, and the supernatant and precipitate were collected by centrifugation. The supernatant was purified with High Affinity Ni-NTA Resin from GenScript. 5 mL of High Affinity Ni-NTA Resin was passed through 5 column volumes of protein buffer, and the supernatant was passed through the column 5 times, and eluted with 10 mL of protein buffer containing 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, and 400 mM imidazole, respectively. The eluate was subjected to SDS-PAGE electrophoresis detection ( Figure 1 The eluate containing the target protein was concentrated using a protein concentration column, aliquoted and quickly frozen in liquid nitrogen, and stored at -80°C.

[0072] (2) Preparation of GINV capsid protein polyclonal antibodies

[0073] ① Select young and middle-aged New Zealand white rabbits weighing about 2.5 kg and mark them. Use GINV capsid protein at a concentration of 1 mg / mL as the immunogen to immunize New Zealand white rabbits. In the first immunization, first mix the antigen completely, and then mix it with Freund's complete adjuvant (paraffin oil: amniotic fluid = 7:1, and add 1 mg / mL inactivated Mycobacterium tuberculosis component) at a volume ratio of 1:1. After complete emulsification, perform multiple subcutaneous injections, 0.2 mL per point, and the immunization dose for each New Zealand white rabbit is 1 mg.

[0074] ② The second to fourth immunizations use Freund's incomplete adjuvant (paraffin oil: amniotic fluid = 7:1), which is still fully mixed with the antigen at a volume ratio of 1:1. The second immunization is performed 14 days after the first immunization. The interval between the second and third immunizations is 7 days, and the immunization dose is the same as the first one.

[0075] ③ On the 7th day after the rabbit was immunized three times, a small sample of serum was collected from the middle ear artery: 200 μL of blood was collected from the ear vein of the immunized New Zealand white rabbit and placed in a sterile centrifuge tube, incubated at 37°C for 2 h, and then kept at 4°C overnight. The next day, centrifuged at 12,000 rpm for 2 min to separate the serum. The indirect ELISA method was used to detect the antibody titer to a level of 1:20,000 or above, and then blood could be collected.

[0076] ④ After 7 days of immunization, fast the immunized New Zealand white rabbits for one day and collect whole blood.

[0077] ⑤ After blood collection, place the blood in a serum separation bottle and incubate at 37℃ for 2 hours, then overnight at 4℃. The next day, collect the precipitated serum with a sterile capillary tube, centrifuge the remaining sample at 3000 rpm for 3 minutes, continue to collect the separated serum, add sodium azide with a mass percentage concentration of 0.02%, and store at -20℃.

[0078] ⑥Crude purification of polyclonal antibody IgG

[0079] Weigh 50g of DEAE-cellulose (DE32 or DE52) and place it in a 1000mL beaker. First, float it with distilled water to remove fine particles. Then, after acid-base treatment, balance it with 0.01-0.05 mol / L phosphate buffer at pH 8.0. Drain the water or filter it with a Buchner filter (with two layers of filter paper) to collect the wet cellulose to reduce the ionic strength. Add 5g of DEAE cellulose to 1mL of serum, stir it thoroughly, and place it at 4℃ for adsorption for 1 hour. The supernatant can be treated again in this way to obtain a relatively pure IgG.

[0080] (3) Horseradish peroxidase labeling of polyclonal antibodies

[0081] ① Dissolve horseradish peroxidase in distilled water, add the newly prepared NaIO4 aqueous solution, mix well, and stand at 4℃ in the dark for 30 min;

[0082] ② Add 0.5 mL of ethylene glycol aqueous solution and incubate at 25°C in the dark for 30 min;

[0083] ③ Add an aqueous solution containing purified antibodies (with an equal molar ratio to horseradish peroxidase), mix well, put into a dialysis bag, and dialyze against 0.05 M, pH 9.6 carbonate buffer for 6 h;

[0084] ④ Add NaBH4 aqueous solution, mix well, and stand at 4℃ in the dark for 2 h;

[0085] ⑤ Slowly add an equal volume of saturated ammonium sulfate solution to the above solution, mix well, let stand at 4°C for 30 min, centrifuge, remove the supernatant, add 20 μL of 0.02 M PBS solution (pH 7.4) to dissolve the precipitate, and dialyze to desalt overnight at 4°C;

[0086] ⑥ Centrifuge and take the supernatant, which is the enzyme-antibody conjugate, add an equal volume of glycerol, and store at -20℃ after packaging.

[0087] (4) Double antibody sandwich enzyme-linked immunosorbent assay for grape berry necrosis virus

[0088] ① Reagent configuration

[0089] a Extraction buffer: 0.87wt% potassium sulfate, 0.5wt% polyvinylpyrrolidone, 0.05wt% Tween-20, 0.1wt% mercaptoethanol, 0.58wt% sodium chloride, 0.2wt% bovine serum albumin, and Tris-HCl buffer (pH 7.4) containing 0.02wt% sodium azide;

[0090] b enzyme buffer, prepared with distilled water, containing 1wt% polyethylene glycol, 0.5wt% bovine serum albumin, 0.01wt% sodium thimerosal, and phosphate buffer (pH 7.4) containing 0.05wt% Tween-20;

[0091] c Washing buffer: phosphate buffer containing 0.05wt% Tween-20, pH 7.2;

[0092] d Color developer A, containing a final concentration of 1.46 wt % sodium dihydrogen phosphate, 0.93 wt % citric acid, and 0.045 wt % hydrogen peroxide aqueous solution;

[0093] e Color developer B, an aqueous solution containing a final concentration of 0.03 wt % tetramethylbenzidine, 0.96 wt % citric acid, 0.019 wt % ethylenediaminetetraethylene glycol sodium salt, 2 wt % DMSO, and 4 wt % glycerol;

[0094] f The stop solution is 2 M H2SO4 solution prepared with distilled water. Grind the grape sample to be tested with extraction buffer and centrifuge it, then aspirate the supernatant;

[0095] g The coating solution was 50 mM carbonate buffer at pH 9.6;

[0096] h blocking solution, phosphate buffer containing 2wt% gelatin and 0.5wt% bovine serum albumin;

[0097] I. Positive quality control, which is obtained by grinding grape leaves that are positive for grape berry necrosis virus RT-PCR with extraction buffer and then centrifuging to obtain the supernatant;

[0098] J Negative quality control material preparation: Grape leaves that are negative for grape berry necrosis virus RT-PCR test are ground with extraction buffer and centrifuged to obtain the supernatant.

[0099] ②Sample treatment: Take 0.1 g of the sample leaf to be tested in a centrifuge tube, add steel beads in turn, freeze quickly in liquid nitrogen, grind into powder with a grinder, add extraction buffer, centrifuge at 4℃, 12 000 rpm for 10 min, and take the supernatant; Coating: Dilute the rabbit anti-polyclonal antibody to a final concentration of 1 μg / mL with coating solution, add 100 μL / well to the microplate, incubate at 37℃ for 2 h, and then overnight at 4℃. The next day, rinse three times with washing solution and spin dry. Blocking: Block with 200 μL / well of blocking solution and incubate at 37℃ for 2 h. Sample addition: Spin off the blocking solution, add the sample to be tested at 100 μL / well, and set up blank control (sample extract), negative control, and positive control at the same time, with the dosage of 100 μL / well, incubate at 37℃ for 30 min, rinse 5 times with washing solution, and spin dry. Add enzyme-labeled antibody: dilute the enzyme-labeled antibody to 0.5 μg / mL with enzyme buffer, 100 μL / well, incubate at 37℃ for 30 min, rinse with washing solution 5 times, and spin dry. Color development: add 10 μL of color developer A and B to each well, incubate at 37℃ in the dark for 30 min. Stop: add 10 μL of stop solution to each well. Detection: measure the OD value of each well at 450 nm on a microplate reader within 10 min.

[0100] In order to verify the precision and accuracy of the kit, we first tested 21 grape leaf samples in the field by RT-PCR. The results are as follows: Figure 2As shown in Figure C, 21 grape leaf samples in the field were tested using the kit of the present invention. Among the 21 samples, 8 samples were negative and 13 samples were positive. The test results of the kit were consistent with the RT-PCR results ( Figure 2 ), the significance analysis showed that there was a very significant difference between the positive sample and the negative sample, and the results were obvious, true and reliable, proving that the kit has high accuracy and sensitivity. Although the present invention has been described in detail above with general descriptions and specific implementation schemes, those skilled in the art may make some modifications or improvements on the basis of the present invention. However, these modifications or improvements made on the basis of not departing from the connotation of the present invention all belong to the scope of protection claimed in the present invention.

Claims

1. A double antibody sandwich enzyme-linked immunosorbent assay kit for grape berry necrosis virus, characterized in that: The kit comprises a GINV-specific polyclonal antibody, which is coated on a polyclonal antibody coating plate. The immunogenic amino acid sequence of the GINV-specific polyclonal antibody is SEQ ID No.

1.

2. The double antibody sandwich ELISA kit for detecting grape berry necrosis virus according to claim 1, characterized in that: The kit also includes horseradish peroxidase-labeled polyclonal antibodies, genetically engineered recombinant GINV coat protein standard products, and quality control products.

3. The double antibody sandwich ELISA kit for grape berry necrosis virus according to claim 1 or 2, characterized in that: The kit also includes auxiliary reagents: extraction reaction solution, washing buffer, enzyme buffer, color developer and reaction termination solution.

4. A method for preparing the grape berry necrosis virus double antibody sandwich enzyme-linked immunosorbent assay kit according to any one of claims 1 to 3, characterized in that: The method includes the steps of preparing a genetically engineered recombinant GINV coat protein standard, preparing a GINV specific polyclonal antibody, preparing a polyclonal antibody coated plate, preparing a horseradish peroxidase labeled polyclonal antibody, preparing a quality control product and preparing an auxiliary reagent.

5. The preparation method according to claim 4, characterized in that: In the preparation step of the GINV coat protein standard, the total RNA of grape leaves infected with GINV is used as a template to amplify the GINV CP gene, clone it into the pET-30a vector to obtain the recombinant plasmid pET30a-GINV-CP, and obtain the 27 kDa coat protein through prokaryotic expression and purification.

6. The preparation method according to claim 5, characterized in that: The nucleotide sequences of the primers used to amplify the CP gene of GINV are as follows: GINV-CP-F: 5'-gatatcggatccgaattcatgtcaattcgtcaggaactgag-3' GINV-CP-R: 5'-ctcgagtgcggccgcaagcttttacatagtaaaagcaccctcgc-3'.

7. The preparation method according to claim 4, characterized in that: In the step of preparing the GINV-specific polyclonal antibody, the GINV-specific polyclonal antibody is obtained by immunizing an animal with the GINV coat protein.

8. The preparation method according to claim 4, characterized in that: In the step of preparing the polyclonal antibody coated plate, the GINV-specific polyclonal antibody obtained by immunizing animals with purified GINV coat protein is coated on an ELISA plate to produce the plate.

9. The preparation method according to claim 4, characterized in that: In the step of preparing the horseradish peroxidase-labeled polyclonal antibody, the GINV-specific polyclonal antibody obtained by immunizing an animal with purified GINV coat protein is labeled with horseradish peroxidase to prepare the antibody.

10. A method for detecting necrosis virus in grape berries, characterized in that: The test kit according to any one of claims 1 to 3 is used to detect grape berry necrosis virus.

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