CN111077318A - Rapid test card for simultaneously detecting TMV, PVY and TVBMV and preparation and use methods thereof - Google Patents
Rapid test card for simultaneously detecting TMV, PVY and TVBMV and preparation and use methods thereof Download PDFInfo
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Abstract
The invention relates to an immune gold-labeled rapid test card which simultaneously detects Tobacco Mosaic Virus (TMV), Potato Virus Y (PVY) and tobacco vein mosaic virus TVBMV in a plant sample by applying the principle of double-antibody sandwich immunochromatography. During detection, firstly adding a sample to be detected into a sample adding hole, if the sample contains a substance to be detected, combining an antigen in the sample with a specific monoclonal antibody marked by colloidal gold on a gold-marked pad in the lateral moving process, and then combining the antigen with a corresponding specific polyclonal antibody on an NC membrane detection line, so that the detection line (T line) develops color, the redundant gold-marked monoclonal antibody is combined with a secondary antibody on a control line, and the control line (C line) develops color; if the sample does not contain the substance to be detected, the detection line (T line) does not develop color, and the control line (C line) develops color. And applying it for rapid, field and sensitive detection of TMV, PVY, TVBMV in leaf samples.
Description
Technical Field
The invention relates to a rapid test card in the technical field of biological engineering and a using method thereof, in particular to a rapid test card for simultaneously detecting Tobacco Mosaic Virus (TMV), Potato Virus Y (PVY) and tobacco vein banding mosaic virus TVBMV and a preparation method and a using method thereof.
Background
Tobacco mosaic virus (hereinafter abbreviated as TMV), potato virus Y (hereinafter abbreviated as PVY), and tobacco vein mosaic virus (hereinafter abbreviated as TVBMV).
At present, the conventional method for detecting TMV, PVY and TVBMV is PCR, needs a special laboratory site, needs special instruments and equipment, has high requirements on operators, needs considerable professional knowledge, skills and operation experience, and has complex pretreatment and long required time in the detection process.
The gold-labeled card detection method based on the lateral flow as the detection principle has been applied in many fields, including food safety detection, plant transgenic detection and the like, but the detection of tobacco viruses is less, and a rapid detection card for simultaneously detecting TMV, PVY, VBMV and the like is not disclosed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a gold-labeled card detection method for simultaneously detecting TMV, PVY and TVBMV, and is applied to quickly, on-site and sensitively detect the TMV, PVY and TVBMV in a blade sample.
The invention is realized by the following technical scheme: a rapid test card for simultaneously detecting tobacco mosaic virus TMV, potato virus Y PVY and tobacco vein banding mosaic virus TVBMV comprises: the detection kit comprises a sample pad, a gold label pad, an NC membrane and an absorption pad, wherein the sample pad, the gold label pad, the NC membrane and the absorption pad are sequentially adhered to a bottom plate, the gold label pad is fixedly provided with a TMV monoclonal antibody, a PVY monoclonal antibody and a TVBMV monoclonal antibody which are marked by colloidal gold, and the NC membrane is provided with three detection lines for respectively fixing TMV, PVY or TVBMV specific polyclonal antibodies.
The TMV monoclonal antibody, the PVY monoclonal antibody and the TVBMV monoclonal antibody are respectively prepared from a hybridoma cell strain secreting the TMV monoclonal antibody, a hybridoma cell strain secreting the PVY monoclonal antibody and a hybridoma cell strain secreting the TVBMV monoclonal antibody.
Fixing 25ng-50ng of three monoclonal antibodies on a gold-labeled pad, wherein the optimized amounts are 30ng of TMV monoclonal antibody, 36ng of PVY monoclonal antibody and 45ng of TVBMV monoclonal antibody; three specific polyclonal antibodies of 0.4-1.2 mug are fixed on the NC film, and the optimized amount is 0.6 mug of TMV polyclonal antibody, 0.9 mug of PVY polyclonal antibody and 0.8 mug of TVBMV polyclonal antibody.
The hybridoma cell strain secreting the TMV monoclonal antibody has a preservation number of CCTCC NO of C2019177; the hybridoma cell strain secreting the PVY monoclonal antibody has a preservation number of CCTCC NO of C2019178; the hybridoma cell strain secreting TVBMV monoclonal antibody has the preservation number of CCTCC NO of C2019180.
Still including closing cap board, closing cap board set up application of sample hole and result observation hole, the application of sample hole corresponds the position of sample pad, the result observation hole corresponds gold mark pad and NC membrane position.
The preparation method of the rapid test card for simultaneously detecting the TMV, PVY and TVBMV comprises the following steps of (1) respectively fixing specific monoclonal antibodies and secondary antibodies of the TMV, PVY and TVBMV on an NC membrane; (2) under alkaline conditions, the colloidal gold particles are respectively combined with the TMV specific monoclonal antibody, the PVY specific monoclonal antibody and the TVBMV specific monoclonal antibody through electrostatic acting force; (3) assembling the rapid test card: the sample pad, the gold label pad, the NC membrane and the absorption pad are sequentially stuck on the bottom plate, and the capped and capped plate and the bottom plate are combined to form the rapid test card.
The using method of the rapid test card for simultaneously detecting the TMV, the PVY and the TVBMV comprises the steps of firstly adding a sample solution into a sample adding hole and horizontally placing the sample solution, observing color change in a result observation hole after 8-10min, and judging; if only the control line C is provided and no corresponding detection line T is provided, the substance to be detected is negative; and if the control line C is provided and the corresponding detection line T is provided, the substance to be detected is positive.
Specifically, a sample to be detected is added into a sample adding hole, if the sample contains a substance to be detected, an antigen in the sample is combined with a specific monoclonal antibody marked by colloidal gold on a gold-marked pad in the lateral moving process, and then is combined with a corresponding specific polyclonal antibody on an NC membrane detection line, so that the detection line (T line) develops color, redundant gold-marked monoclonal antibodies are combined with a secondary antibody on a control line, and the control line (C line) develops color; if the sample does not contain the substance to be detected, the detection line (T line) does not develop color, and the control line (C line) develops color.
The TMV polyclonal antibody, the PVY polyclonal antibody and the TVBMV polyclonal antibody are respectively obtained by purifying serum taken from an experimental rabbit immunized by TMV, PVY and TVBMV virus particles.
The novel TMV/PVY/TVBMV three-in-one rapid test card prepared by the invention has better anti-interference effect on tobacco leaves, and can be widely applied to rapid detection of tobacco. Compared with the operation of the existing speed measuring card, the speed measuring card has the following specific advantages.
(1) TMV, PVY and TVBMV can be detected simultaneously on the same card, but not the simple physical glue of three single cards, and the application of sample only needs 1 time, and the reaction is all gone on under same condition moreover.
(2) The pretreatment of the sample is completely unified, the operation is simple, and the sample loading amount of 100 microliter of sample is only needed.
(3) Short detection time (1-5 min) and uniform result judgment standard. A complete negative result (only 1C-line appeared); positive (+) position: so long as any one T line of TMV/PVY/TVBMV develops color.
(4) The technical defect that a macromolecular compound cannot be accurately detected by adopting a monoclonal antibody detection method in the prior art can be overcome by adopting the principle of double-antibody sandwich immunochromatography (namely, antibodies are fixed at a gold-labeled pad and a detection line, and antigens are fixed at the detection line in the prior art).
The three-in-one immune colloidal gold rapid test card for simultaneously testing TMV, PVY and TVBMV provided by the invention can provide a rapid, on-site and sensitive test method for practical test departments, and is mainly applied to the test of leaf samples.
Description of the drawings:
FIG. 1 is a schematic structural view of the present invention; 1-result observation well; 2-C line, control line; 3-TMV line; 4-PVY line; 5-TVBMV line; 6-well for application.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will be described in detail with reference to specific examples.
(1) Preparation of TMV, PVY and TVBMV monoclonal antibody
Taking a TMV monoclonal antibody hybridoma cell strain with the preservation number of CCTCC NO of C2019177, a PVY monoclonal antibody hybridoma cell strain with the preservation number of CCTCC NO of C2019178 and a TVBMV monoclonal antibody hybridoma cell strain with the preservation number of CCTCC NO of C2019180, and carrying out expanded culture in a cell culture bottle;
injecting liquid paraffin into the abdominal cavity of BALB/c mice of 6-8 weeks old, wherein each mouse is 0.2 mL. After 10 days, a certain number of monoclonal antibody hybridoma cells were intraperitoneally inoculated. The abdomen of the mouse is obviously expanded, ascites is collected by an elbow dropper, and the titer of ascites antibody is measured by indirect ELISA.
Centrifuging ascites of mouse for 15min (2000 rpm, room temperature), removing upper oil, adding saturated ammonium sulfate dropwise slowly at 4 deg.C under stirring to half-saturation, stirring for 30min, centrifuging for 30min (13000 rpm, 4 deg.C), and removing supernatant; the pellet was dissolved in an appropriate amount of PBS (0.01M pH7.4); slowly adding saturated ammonium sulfate dropwise to 33% under stirring at 4 deg.C, stirring for 30min, centrifuging for 30min (13000 rpm, 4 deg.C), and removing supernatant; the pellet was dissolved in an appropriate amount of PBS (0.01M, pH7.4), dialyzed overnight at 4 ℃ to determine the protein content, and frozen at-20 ℃ for use.
Purifying with Protein G column after ammonium sulfate precipitation, passing 5ml ultrapure water through the new column, and balancing the purified column with 5ml 0.4M PB buffer solution (pH 7.0); the antibody passes through the column slowly in the process, so that the antibody protein is better combined on the binding site; the purification column was equilibrated with 10 ml0.4m PB buffer (pH 7.0); eluting the antibody on the binding site with 5ml0.1M glycine-hydrochloric acid buffer (pH 7.0), and adding 1M Tris-HCI (pH2.7) to neutralize glycine, so that the pH is kept neutral suitable for antibody preservation; equilibrating the purification cartridge with 10 ml0.4m PB buffer (pH 7.0); 5mL of 20% ethanol solution was passed through the column, and the purified column was stored at 4 ℃.
(2) Preparation of rabbit polyclonal antibody
One 2-month-old female New Zealand white rabbit is immunized for three times, and the inguinal subcutaneous multipoint injection is adopted. Each new Zealand white rabbit antigen is immunized with 500 μ g of antigen, the first immunization is emulsified with equivalent Freund's complete adjuvant, the second immunization is emulsified with equivalent Freund's incomplete adjuvant, the third immunization is mixed with equivalent physiological saline, and the mixture is injected into ear vein to strengthen the immunization. Blood was taken 14 days after the booster immunization.
Centrifuging serum for 15min (4000 rpm, room temperature), collecting supernatant, adding saturated ammonium sulfate dropwise slowly at 4 deg.C under stirring to half saturation, stirring for 30min, centrifuging for 30min (13000 rpm, 4 deg.C), and discarding supernatant; the pellet was dissolved in an appropriate amount of PBS (0.01M, pH 7.4); slowly adding saturated ammonium sulfate dropwise to 33% under stirring at 4 deg.C, stirring for 30min, centrifuging for 30min (13000 rpm, 4 deg.C), and removing supernatant; the precipitate was dissolved in an appropriate amount of PBS (0.01M, pH7.4), dialyzed overnight at 4 ℃ and assayed for antibody content, and frozen at-20 ℃ for future use. Ammonium sulfate precipitation, purifying with Protein G column, passing 5ml ultrapure water through the new column, and balancing the purified column with 5ml 0.4M PB buffer solution (pH 7.0); the antibody passes through the column slowly in the process, so that the antibody protein is better combined on the binding site; the column was equilibrated with 10 ml of 0.4M PB buffer (pH 7.0); the antibody on the binding site was eluted with 5ml of 0.1M glycine-hydrochloric acid buffer (pH2.7), and glycine was neutralized by adding 1M Tris-HCl (pH 8.0) to maintain the pH at neutrality suitable for antibody preservation.
(3) Production of TMV/PVY/TVBMV three-in-one quick test card
1) The cover plate (plastic plate) is provided with two holes, namely a sample adding hole and an observation hole, wherein the size of the sample adding hole is 3mm multiplied by 7mm, and the size of the observation hole is 4mm multiplied by 18 mm; the sample adding hole corresponds to the position of the sample pad, and the result observation hole corresponds to the positions of the gold label pad and the NC film;
2) a sample pad (glass fiber film), a test strip fixed with colloidal gold labeled antibody, an NC film and an absorption pad are sequentially adhered on a bottom plate (plastic plate). The size of the glass fiber membrane is 4mm multiplied by 15 mm; the size of the gold-labeled antibody test strip is 3mm multiplied by 4 mm; the size of the NC membrane is 4mm multiplied by 28 mm; the size of the absorption pad is 4mm multiplied by 19 mm;
3) with HAuCl4Preparing colloidal gold with the particle size of 25nm by adopting a sodium citrate reduction method, and respectively combining the colloidal gold particles with a TMV specific monoclonal antibody, a PVY specific monoclonal antibody and a TVBMV specific monoclonal antibody under an alkaline condition through electrostatic acting force;
4) fixing 25ng-50ng of three monoclonal antibodies on a gold label pad by using a film-scratching gold-spraying marking machine, wherein the optimized amounts are 30ng of TMV monoclonal antibody, 36ng of PVY monoclonal antibody and 45ng of TVBMV monoclonal antibody; three specific polyclonal antibodies of 0.4-1.2 mug are fixed on the NC film, the optimized amount is 0.6 mug of TMV polyclonal antibody, 0.9 mug of PVY polyclonal antibody and 0.8 mug of TVBMV polyclonal antibody;
5) respectively fixing specific polyclonal antibodies and secondary antibodies (goat anti-mouse) of TMV, PVY and TVBMV on an NC membrane by using a membrane-cutting gold-spraying marking machine, naturally drying, sealing in a sealing buffer solution for one hour, washing in a washing buffer solution for two times, and naturally drying at room temperature; the second antibody (goat anti mouse) on the control line C is an antibody which is prepared by taking an antibody as an antigen and can recognize immunoglobulin IgG of the antibody, and the antibody can react when passing through the second antibody, so that the second antibody can be used as a quality control line.
6) The capped sealing cover plate and the bottom plate are combined to form a rapid test card; the quick test card is dried at 37 deg.C for 30 min.
(3) Evaluation of virus particle detection effect of TMV/PVY/TVBMV three-in-one rapid test card
① adding 100 μ L blank sample solution (pH 7.4, 0.2mol/L PBS: 8g sodium chloride, 3.35 g disodium hydrogen phosphate dodecahydrate, 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride, double distilled water dissolved to constant volume to 1L) into the sample adding hole, reacting for 8 minutes at room temperature, observing color development result in the observation hole, the result shows that the control line C line in the observation hole is wine red, and the detection lines T1 and T2 are not color development.
② adding 100 μ L of 1 μ g/ml TMV virion solution (diluted with PBS solution as above) into the well, and following the same procedure as the blank sample, the control line C and the detection line T1 in the observation well appeared wine red, and the detection lines T2 and T3 appeared colorless.
③ mu.L of PVY virus particle solution (prepared by diluting with PBS solution as above) of 1. mu.g/ml is added into the sample adding hole, and the same operation steps as the blank sample are carried out, the results show that the control line C and the detection line T2 in the observation hole are wine red, and the detection lines T1 and T3 are colorless.
④ mu.L of 1. mu.g/ml TVBMV virion solution (diluted with PBS solution as above) was added to the wells, and the same procedure as the blank samples was followed, indicating that the control line C and the detection line T3 in the wells appeared wine red, and the detection lines T1 and T2 were colorless.
⑤ mu.L of solution containing 1 mu g/ml of TMV virus particles and 1 mu g/ml of PVY virus particles (prepared by diluting with the PBS solution) is added into the sample adding hole, and the same operation steps as the blank sample are carried out, wherein the results show that the control line C line, the detection lines T1 and T2 lines in the observation hole are wine red, and the detection line T3 line is colorless.
⑥ mu.L of a solution containing 1 mu g/ml of TMV virus particles and 1 mu g/ml of TVBMV virus particles (prepared by diluting with the PBS solution) is added into the sample adding hole, and the same operation steps as the blank sample are carried out, the results show that the control line C line, the detection lines T1 and T3 lines in the observation hole are wine red, and the detection line T2 line is colorless.
⑦ mu.L of a solution containing both 1. mu.g/ml of PVY virus particles and 1. mu.g/ml of TVBMV virus particles (diluted with the same PBS solution) was added to the wells, and the same procedure as for the blank samples was followed, it was found that the control line C, the detection lines T2 and T3 in the wells appeared wine red, and the detection line T1 was colorless.
⑧ mu.L of a solution containing 1. mu.g/ml of TMV virus particles, 1. mu.g/ml of PVY virus particles and 1. mu.g/ml of TVBMV virus particles (diluted with the same PBS solution as above) was added to the wells, and the same procedure as that for the blank samples was followed, the control line C, the detection lines T1, T2 and T3 in the wells were shown to be wine red.
(5) Blade sample detection effect evaluation of TMV/PVY/TVBMV three-in-one speed measuring card
① mu.L of the negative leaf sample extract was added to the wells and the same procedure was followed as for the blank samples, indicating that the control line C in the wells appeared wine red and the detection lines T1 and T2 did not appear.
② mu.L of TMV positive leaf sample extract was added to the wells, and the same procedure was followed as for the blank samples, indicating that the control line C and the detection line T1 in the wells appeared wine red, and the detection lines T2 and T3 were colorless.
③ adding 100 μ L of PVY positive leaf sample extractive solution into the well, and performing the same operation as the blank sample to obtain the control line C and the detection line T2 in the observation well showing wine red color, and the detection lines T1 and T3 showing no color.
④ and adding 100 mu L of TVBMV positive leaf sample extracting solution into the sample adding hole, and according to the same operation steps as the blank sample, the result shows that a control line C and a detection line T3 in the observation hole are wine red, and detection lines T1 and T2 are colorless.
⑤ and adding 100 mu L of leaf sample extracting solution with positive TMV and PVY into the sample adding hole, and according to the same operation steps as the blank sample, the results show that the control line C line, the detection line T1 and the detection line T2 line in the observation hole are wine red, and the detection line T3 line is colorless.
⑥ and adding 100 mu L of leaf sample extracting solution with positive TMV and TVBMV into the sample adding hole, and according to the same operation steps as the blank sample, the result shows that a control line C line, a detection line T1 and a detection line T3 line in the observation hole are wine red, and a detection line T2 line is colorless.
⑦ and adding 100 mu L of leaf sample extracting solution with positive PVY and TVBMV into the sample adding hole, and according to the same operation steps as the blank sample, the result shows that a control line C line, a detection line T2 and a detection line T3 line in the observation hole are wine red, and a detection line T1 line is colorless.
⑧ mu.L of leaf sample extract positive to TMV, PVY and TVBMV was added to the wells, and the same procedure was followed as for the blank samples.
The sensitivity is obtained through tests: TMV: 200ppb, PVY: 500ppb, TVBMV: 500 ppb.
The applicant states that the present invention patent is described by the above embodiments, and it should be understood by those skilled in the art that any modification of the present invention is within the protection scope of the present invention.
Claims (8)
1. A rapid test card for simultaneously detecting tobacco mosaic virus TMV, potato virus Y PVY and tobacco vein banding mosaic virus TVBMV comprises: sample pad, gold mark pad, NC membrane and the absorption pad of pasting in proper order on the bottom plate, its characterized in that: the gold-labeled pad is fixed with a TMV monoclonal antibody, a PVY monoclonal antibody and a TVBMV monoclonal antibody which are labeled by colloidal gold, and the NC membrane is provided with three detection lines for respectively fixing TMV, PVY or TVBMV specific polyclonal antibodies.
2. The rapid test card for simultaneously detecting tobacco mosaic virus TMV, potato virus Y PVY and tobacco vein banding mosaic virus TVBMV according to claim 1, wherein: the TMV monoclonal antibody, the PVY monoclonal antibody and the TVBMV monoclonal antibody are respectively prepared from a hybridoma cell strain secreting the TMV monoclonal antibody, a hybridoma cell strain secreting the PVY monoclonal antibody and a hybridoma cell strain secreting the TVBMV monoclonal antibody.
3. The rapid test card for simultaneously detecting tobacco mosaic virus TMV, potato virus Y PVY and tobacco vein banding mosaic virus TVBMV according to claim 1, wherein: fixing 25ng-50ng of three monoclonal antibodies on a gold-labeled pad, wherein the optimized amounts are 30ng of TMV monoclonal antibody, 36ng of PVY monoclonal antibody and 45ng of TVBMV monoclonal antibody; three specific polyclonal antibodies of 0.4-1.2 mug are fixed on the NC film, and the optimized amount is 0.6 mug of TMV polyclonal antibody, 0.9 mug of PVY polyclonal antibody and 0.8 mug of TVBMV polyclonal antibody.
4. The rapid test card for simultaneously detecting tobacco mosaic virus TMV, potato virus Y PVY and tobacco vein banding mosaic virus TVBMV according to claim 2, wherein: the hybridoma cell strain secreting the TMV monoclonal antibody has a preservation number of CCTCC NO of C2019177; the hybridoma cell strain secreting the PVY monoclonal antibody has a preservation number of CCTCC NO of C2019178; the hybridoma cell strain secreting TVBMV monoclonal antibody has the preservation number of CCTCC NO of C2019180.
5. The rapid test card for simultaneously detecting tobacco mosaic virus TMV, potato virus Y PVY and tobacco vein banding mosaic virus TVBMV according to claim 1, wherein: still including closing cap board, closing cap board set up application of sample hole and result observation hole, the application of sample hole corresponds the position of sample pad, the result observation hole corresponds gold mark pad and NC membrane position.
6. The method for preparing a rapid test card for simultaneously detecting TMV, PVY and TVBMV as claimed in any one of claims 1 to 5, wherein the method comprises the following steps: (1) fixing specific monoclonal antibodies and secondary antibodies of TMV, PVY and TVBMV on an NC membrane respectively; (2) under alkaline conditions, the colloidal gold particles are respectively combined with the TMV specific monoclonal antibody, the PVY specific monoclonal antibody and the TVBMV specific monoclonal antibody through electrostatic acting force; (3) assembling the rapid test card: the sample pad, the gold label pad, the NC membrane and the absorption pad are sequentially stuck on the bottom plate, and the capped and capped plate and the bottom plate are combined to form the rapid test card.
7. The use of the rapid test card for simultaneously detecting TMV, PVY and TVBMV according to any one of claims 1 to 5, wherein the rapid test card comprises: firstly, adding a sample solution into a sample adding hole and horizontally placing, observing color change in a result observation hole after 8-10min, and judging; if only the control line C is provided and no corresponding detection line T is provided, the substance to be detected is negative; and if the control line C is provided and the corresponding detection line T is provided, the substance to be detected is positive.
8. The rapid test card for simultaneously detecting TMV, PVY and TVBMV according to any one of claims 1 to 4, wherein the card comprises: the TMV polyclonal antibody, the PVY polyclonal antibody and the VBMV polyclonal antibody are respectively obtained by purifying serum taken from an experimental rabbit immunized by TMV, PVY and TVBMV virus particles.
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| 纪玲玲 等: "烟草脉带花叶病毒试纸条的制作及田间病株的快速检测", 《西北农业学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112557656A (en) * | 2021-01-19 | 2021-03-26 | 黑龙江省农业科学院克山分院 | Test strip for synchronously detecting various potato viruses and preparation method and application thereof |
| CN112557656B (en) * | 2021-01-19 | 2023-09-05 | 黑龙江省农业科学院克山分院 | Test strip for synchronously detecting various potato viruses and preparation method and application thereof |
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