CN111596063A - Rapid test card for simultaneously detecting PVY and TVBMV and preparation and use methods thereof - Google Patents
Rapid test card for simultaneously detecting PVY and TVBMV and preparation and use methods thereof Download PDFInfo
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- CN111596063A CN111596063A CN201911424656.5A CN201911424656A CN111596063A CN 111596063 A CN111596063 A CN 111596063A CN 201911424656 A CN201911424656 A CN 201911424656A CN 111596063 A CN111596063 A CN 111596063A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention relates to an immune gold-labeled rapid test card which simultaneously detects Potato Virus Y (PVY) and Tobacco Vein Banding 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 two viruses or one of the two viruses, the viruses can be combined with the colloidal gold-labeled specific monoclonal antibody on the gold-labeled pad in the process of sample chromatographic movement, then the viruses are deposited by the antibody on the corresponding virus detection line (T line) of the NC membrane, the detection line (T line) of the corresponding viruses develops color, the redundant gold-labeled monoclonal antibody is deposited by the secondary antibody on the control line in the process of continuous movement, and the control line (C line) develops color; if the sample does not contain the two viruses, the two detection lines (T lines) do not develop color, and the control line (C line) develops color. The rapid detection card can be used for accurately, rapidly and sensitively detecting PVY and TVBMV in various samples on site.
Description
Technical Field
The invention relates to a rapid test card in the technical field of bioengineering and a use method thereof, in particular to a rapid test card for simultaneously detecting Potato Virus Y (PVY) and Tobacco Vein Banding Mosaic Virus (TVBMV) and a preparation method and a use method thereof.
Background
Potyvirus (hereinafter referred to as PVY) of Potyviridae (PVY)Potyviridae) Genus potyvirus (A), (B), (C), (Potyvirus) A virus. The virions are linear and the genome is positive single-stranded RNA. The PVY has a plurality of strains, wherein the N strain can cause the vein necrosis of the tobacco and other host plants, and can cause the crop failure in severe cases.
Tobacco vein banding mosaic virus (TVBMV for short), a potyviridae (Potyviridae) Genus potyvirus (A), (B), (C), (Potyvirus) A virus. The virions are linear and the genome is positive single-stranded RNA. The TVBMV diseases are very extensive, but the symptoms are easy to be confused with other viruses, and the situation that the TVBMV is misdiagnosed during production and an improper prevention method is adopted frequently occurs.
At present, the conventional method for detecting PVY and TVBMV is PCR, needs a special laboratory field, 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 chromatography flow as the detection principle has been applied in many fields, including food safety detection, plant transgenic detection and the like, but the detection for tobacco viruses is less, and the rapid detection card for simultaneously detecting PVY, TVBMV and the like is not reported in the open.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a gold-labeled card detection method for simultaneously detecting PVY and TVBMV, and the gold-labeled card detection method is applied to quickly, on-site and sensitively detect the PVY and TVBMV in a leaf sample.
The invention is realized by the following technical scheme: a rapid test card for simultaneously detecting potato virus Y PVY and tobacco vein banding mosaic virus TVBMV comprises: the sample pad, the gold label pad, the NC membrane and the absorption pad are sequentially adhered to the bottom plate, the gold label pad is fixed with a colloidal gold-labeled PVY monoclonal antibody and a TSWV monoclonal antibody, and the NC membrane is provided with two detection lines for respectively fixing PVY or TVBMV specific polyclonal antibodies.
The PVY monoclonal antibody and the TVBMV monoclonal antibody are respectively prepared from a hybridoma cell strain secreting the PVY monoclonal antibody and a hybridoma cell strain secreting the TVBMV monoclonal antibody.
The amount of each monoclonal antibody on the gold label pad is 5ng-50ng, the optimized amount is 24ng of PVY monoclonal antibody and 36ng of TVBMV monoclonal antibody; the amount of polyclonal antibody on NC membrane is 0.4-1.2. mu.g, the optimized amount is 0.4. mu.g PVY polyclonal antibody and 0.45. mu.g TVBMV polyclonal antibody.
The hybridoma cell strain secreting the PVY monoclonal antibody has a preservation number of CCTCC NO of C2019178, and the preservation unit is: china center for type culture Collection, collection address: wuhan, Wuhan university, preservation date: year 2019, month 9, day 27, category name: hybridoma cell line FL 204-46; the hybridoma cell strain secreting TVBMV monoclonal antibody has the preservation number of CCTCC NO of C2019180, and the preservation unit: china center for type culture Collection, collection address: wuhan, Wuhan university, preservation date: year 2019, month 9, day 27, category name: hybridoma cell line FL 349-11.
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 potato virus Y PVY and the tobacco vein banding mosaic virus TVBMV comprises the following steps of (1) respectively fixing specific polyclonal antibodies and secondary antibodies of the PVY and the TVBMV on an NC membrane; (2) respectively combining the colloidal gold particles with the PVY-resistant specific monoclonal antibody and the TVBMV-resistant specific monoclonal antibody under an alkaline condition 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 potato virus Y PVY and the tobacco vein banding mosaic virus TVBMV comprises the steps of firstly adding a sample solution into a sample adding hole and horizontally placing the sample solution, and observing color change in a result observation hole after 1min to make judgment; 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.
The PVY/TVBMV two-in-one rapid test card prepared by the method has the following concrete steps of rapidly testing an actual blade sample: first, 100. mu.L of the sample solution was added to the well and placed horizontally. The sample solution will permeate to the test strip of colloidal gold labeled antibody along the sample pad and compete with the specific antigen on the NC membrane to bind the specific monoclonal antibody immobilized on the test strip. After 1min, the color change was observed in the observation holes to make a judgment.
And (5) judging a result: a complete negative result (1 line C present); positive (+) position: so long as any one of the PVY/TVBMV T lines develops color.
The novel PVY/TVBMV two-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) PVY and TVBMV can be detected simultaneously on the same card, rather than the simple physical glue of two single cards, 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) The detection time is short (1 min), and the result judgment standards are unified. A complete negative result (only 1C-line appeared); positive (+) position: so long as any one of the PVY/TVBMV T lines 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 two-in-one immune colloidal gold rapid test card for simultaneously testing 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 view of the construction of the instant card of the present invention, 1-result observation hole; 2-C line, control line; 3-PVY line; 4-TVBMV line; 5-well for application.
Detailed Description
The following examples are provided in connection with the present disclosure:
(1) preparation of PVY and TVBMV monoclonal antibody
Taking a PVY monoclonal antibody hybridoma cell line with the preservation number of C2019178 and a TVBMV monoclonal antibody hybridoma cell line with the preservation number of C2019180, and carrying out expanded culture in a cell culture bottle;
injecting liquid paraffin into the abdominal cavity of a BALB/c mouse with the age of 7 weeks, 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) Manufacture of PVY/TVBMV two-in-one speed measuring 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 PVY specific monoclonal antibody and a TVBMV specific monoclonal antibody under an alkaline condition through electrostatic acting force;
4) fixing two kinds of monoclonal antibodies of 5ng-50ng on a gold label pad by using a membrane-scribing gold-spraying marking machine, wherein the optimized amount is 24ng of PVY monoclonal antibody, 36ng of TVBMV monoclonal antibody, 0.4 mu g-1.2 mu g of two kinds of polyclonal antibodies are fixed on an NC membrane, and the optimized amount is 0.4 mu g of polyclonal antibody PVY and 0.45 mu g of TVBMV polyclonal antibody;
5) fixing specific polyclonal antibodies and secondary antibodies (goat anti-mouse) of PVY and TVBMV on an NC membrane respectively by using a membrane-scribing 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.
(4) Evaluation of virus particle detection effect of PVY/TVBMV two-in-one rapid test card
mu.L of a blank sample solution (pH 7.4, 0.2mol/L PBS: 8g of sodium chloride, 3.35 g of disodium hydrogenphosphate dodecahydrate, 0.2g of potassium dihydrogenphosphate, 0.2g of potassium chloride, and a volume of 1L dissolved in double distilled water) was added to the wells, reacted at room temperature for 8 minutes, and then the color development was observed in the observation wells. The results indicated that the control line C, T1 and T2, which exhibited a distinct wine-red color in the viewing port, did not develop color.
② 100 mu L of 1 mu g/ml PVY virus particle solution (prepared by diluting 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 observation hole is provided with a C line, a T1 line and a T2 line which are colorless.
③ adding 100 μ L of 1 μ g/ml TVBMV virion solution (prepared by diluting with PBS solution as above) into the sample adding hole, and performing the same operation as the blank sample. The results show that the observation hole is provided with a C line, a T2 line and a T1 line which are colorless.
And fourthly, adding 100 mu L of solution (prepared by diluting the PBS solution as the sample) containing 1 mu g/ml of PVY virus particles and 1 mu g/ml of TVBMV virus particles into the sample adding hole, and performing the same operation steps as the blank sample. The result shows that the control line C line, the detection line T1 line and the detection line T2 line in the observation hole are wine red.
(5) Blade sample detection effect evaluation of PVY/TVBMV two-in-one speed measurement card
First, 100. mu.L of the negative leaf sample extract was added to the wells, and the same procedure as for the blank samples was followed. The results show that the control line C, the detection line T1 and the detection line T2, which are obviously wine red, do not develop color in the observation hole.
② adding 100 mul of PVY positive leaf sample extracting solution into the sample adding hole, and carrying out the same operation steps as the blank sample. The result shows that the control line C line and the detection line T1 line in the observation hole are wine red, and the detection line T2 line is colorless.
And thirdly, adding 100 mu L of TVBMV positive leaf sample extracting solution into the sample adding hole, and performing the same operation steps as the blank sample. The result shows that the control line C line and the detection line T2 line in the observation hole are wine red, and the detection line T1 line is colorless.
And fourthly, adding 100 mu L of leaf sample extracting solution with positive PVY and TVBMV into the sample adding hole, and performing the same operation steps as the blank sample. The result shows that the control line C line, the detection line T2 line and the detection line T1 line in the observation hole are wine red.
The detection sensitivity of the detection card is obtained through tests: PVY: 300ppb, 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 (7)
1. A rapid test card for simultaneously detecting 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 colloidal gold-labeled PVY monoclonal antibody and a TVBMV monoclonal antibody, and the NC membrane is provided with two detection lines for respectively fixing PVY or TVBMV specific polyclonal antibodies.
2. The rapid test card for simultaneously detecting potato virus Y PVY and tobacco vein banding mosaic virus TVBMV according to claim 1, wherein: the PVY monoclonal antibody and the TVBMV monoclonal antibody are respectively prepared from 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 potato virus Y PVY and tobacco vein banding mosaic virus TVBMV according to claim 1, wherein: the amount of each monoclonal antibody on the gold label pad is 5ng-50ng, the optimized amount is 24ng of PVY monoclonal antibody and 36ng of TVBMV monoclonal antibody; the amount of specific polyclonal antibody on NC membrane is 0.4-1.2. mu.g, the optimized amount is 0.4. mu.g PVY polyclonal antibody and 0.45. mu.g TVBMV polyclonal antibody.
4. The rapid test card for simultaneously detecting potato virus Y PVY and tobacco vein banding mosaic virus TVBMV according to claim 2, wherein: 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 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 potato virus Y PVY and tobacco vein banding mosaic virus TVBMV according to any one of claims 1 to 5, wherein the method comprises the following steps: (1) respectively fixing specific polyclonal antibodies and secondary antibodies of PVY and TVBMV on an NC membrane; (2) respectively combining the colloidal gold particles with the PVY-resistant specific monoclonal antibody and the TVBMV-resistant specific monoclonal antibody under an alkaline condition 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 the potato virus Y PVY and the tobacco vein banding mosaic virus TVBMV according to any one of claims 1 to 5, wherein: firstly, adding a sample solution into an adding hole and horizontally placing, and observing color change in a result observation hole after 1min to make judgment; 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.
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| CN201911424656.5A CN111596063A (en) | 2019-12-31 | 2019-12-31 | Rapid test card for simultaneously detecting PVY and TVBMV and preparation and use methods thereof |
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| CN201911424656.5A CN111596063A (en) | 2019-12-31 | 2019-12-31 | Rapid test card for simultaneously detecting PVY and TVBMV and preparation and use methods thereof |
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| CN111596063A true CN111596063A (en) | 2020-08-28 |
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