CN110297088A - Foot and mouth disease virus quantitative testing test paper card and preparation method thereof - Google Patents
Foot and mouth disease virus quantitative testing test paper card and preparation method thereof Download PDFInfo
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- CN110297088A CN110297088A CN201910646237.XA CN201910646237A CN110297088A CN 110297088 A CN110297088 A CN 110297088A CN 201910646237 A CN201910646237 A CN 201910646237A CN 110297088 A CN110297088 A CN 110297088A
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/558—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
Abstract
The present invention relates to foot and mouth disease virus quantitative testing test paper cards and preparation method thereof, belong to foot and mouth disease virus rapid detection technical field.Test card of the present invention include get stuck, PVC liner plate, absorption pad, nitrocellulose filter, gold-labelled pad and sample pad, indicate O-shaped rabbit-anti, A type or Asia I type antibodies against foot-and-mouth disease virus in the gold-labelled pad；Coating indicates the quality control band of goat anti-rabbit igg antibody and indicates the detection band of O-shaped rabbit-anti, A type or Asia I type antibodies against foot-and-mouth disease virus on the nitrocellulose filter.Test card of the present invention can determine O-shaped aftosa, A type or Asia I type foot-and-mouth disease virus and its content, and easy to operate quick, be suitable for the detection and evaluation of vaccine quality.
Description
Technical field
The present invention relates to foot and mouth disease virus rapid detection technical fields, and in particular to foot and mouth disease virus quantitative testing test paper card
And preparation method thereof.
Background technique
Aftosa (Foot andMouth Disease, FMD) is the artiodactyls as caused by foot and mouth disease virus (FMDV)
Acute, the hot, contagious disease suffered from altogether.Foot and mouth disease virus has A, O, C, Asia1, SAT1, SAT2 and SAT3 totally 7 serum
Type.Although morbidity sign caused by various virus is identical, can still be infected between type without cross immunity, after being ill rehabilitation or immune animal
Other serotypes and fall ill.Therefore, the same area can multiple serotypes popular simultaneously aftosa.Aftosa is mainly endangered
The major livestocks poultry kind such as evil pig, cattle and sheep, can cause huge economic loss.Chinese Pigs, ox, sheep cultivation amount is maximum, neighbouring country is numerous,
Diagnosis and prevention and control aftosa not only play a crucial role to national Development of farming and animal husbandry, but also have important meaning to global aftosa prevention and control
Justice.Since aftosa is propagated rapidly, it is difficult to prevent and treat, remedial measure is few, so quickly sensitive detection technique for taking in time
Measure, the sprawling of prevention and control epidemic situation are most important.
Vaccine immunity is the main means of prevention and control aftosa, and inactivated foot-and-mouth disease vaccine is main vaccine used at present,
Important function has been played in prevention and control.The content of Effective Antigens and complete virion in vaccine is inactivated foot-and-mouth disease vaccine matter
The key of amount.Therefore, the detection of inactivated foot-and-mouth disease vaccine viral level, can control in the production process of inactivated foot-and-mouth disease vaccine
Quality, compare the difference of vaccine between each batch, evaluate vaccine semi-finished product, final product quality, to improve production efficiency, improve life
Production. art.When carrying out aftosa immunity inoculation to animal, often between different manufacturers or different batches there are the difference of vaccine valence,
Low liter vaccine easily leads to immuning failure, and therefore, viral level quickly, in Accurate Determining foot and mouth disease virus inactivated vaccine is right
Effective aftosa epidemic preventing vaccine is screened to have practical application value.Before inactivated foot-and-mouth disease vaccine immunity inoculation, quickly comment
Viral level in valence vaccine is of great significance to immunoprophylaxis work.
Currently, the method for detection foot and mouth disease virus is mainly virus purification, serology and molecular biological variety identification method, this
Three classes method needs professional technology or equipment or detection time relatively long mostly.Foot and mouth disease virus assay is main
The method of application is mainly the methods of density gradient method, gel filtration chromatography, ELISA, fluorescence quantitative RT-RCR, sucrose density
There is no limit for serotype strain of the gradient centrifugation to FMDV, but can not detect the antigen of each serotype of multivalence finished product vaccine
Content；ELISA method operation is relatively easy, disposably can detect a large amount of samples, but the process for preparing monoclonal antibody is complicated,
And it is restricted to the Strain of detection；Gel filtration chromatography then high degree of automation, but detection sensitivity and impurity is contained
Measure that higher sample separative efficiency is not high at present, fluorescence quantitative RT-RCR is often due to the influence of the factors such as reagent, operation can produce
Raw some errors, and limited by investigative technique level, the precision of absolute quantitation and accuracy be not high.Therefore a kind of behaviour is established
Make quickly and easily, as a result effectively accurately foot and mouth disease virus quantitative detecting method is of great significance.
Summary of the invention
The purpose of the present invention is to provide foot and mouth disease virus quantitative testing test paper cards and preparation method thereof.Examination of the present invention
Paper card can determine O-shaped aftosa, A type or Asia I type foot-and-mouth disease virus and its content, and easy to operate quick, be suitable for
The detection and evaluation of vaccine quality.
The present invention provides foot and mouth disease virus quantitative testing test paper cards, it is preferred that the sample packet of the test card detection
Include aftosa vaccine semi-finished product, aftosa vaccine finished product, the tissue containing foot and mouth disease virus or the fluid sample containing foot and mouth disease virus.
Preferably, the aftosa vaccine finished product carries out demulsification processing before detection；The group containing foot and mouth disease virus
Decentralized processing is carried out before being woven in detection；The fluid sample containing foot and mouth disease virus is centrifuged before detection, takes supernatant.
Preferably, demulsification processing are as follows: by aftosa vaccine finished product and demulsifier according to mass ratio be (4~5): 1
Mixing, 3000~5000rpm is centrifuged 10~15min after oscillation, takes lower liquid；
The demulsifier is n-butanol, n-amyl alcohol or trichloro ethylene.
Preferably, the decentralized processing are as follows: the tissue containing foot and mouth disease virus is ground, then with dispersing agent according to
Mass ratio is 1:5 mixing, and overnight, next day is centrifuged 5~10min with 3000~5000rpm to 4 DEG C of leaching poison, takes supernatant；
The dispersing agent is 0.11mol/L phosphate buffer or DMEM culture solution.
The present invention also provides the preparation methods of foot and mouth disease virus quantitative testing test paper card described in above-mentioned technical proposal, including
Following steps:
1) pH to 8.0~8.5 for adjusting colloidal gold solution, adds final concentration of 2~15 μ g/ml O-shaped, A type or Asia I type
Antibodies against foot-and-mouth disease virus, stir 30~40min, be added final concentration of 0.5~1% casein, stir 30~40min, 8000
~10000rpm is centrifuged 30~60min, discards supernatant, and gained precipitating is redissolved in the colloid of colloidal gold solution volume 1/10~1/20
In golden buffer, immune colloid gold solution is obtained, the immune colloid gold solution is sprayed, obtains indicating that rabbit-anti is O-shaped, A
The gold-labelled pad of type or Asia I type foot-and-mouth disease antiviral antibody；
2) phosphate buffer is used, is diluted to 0.2~1mg/ for goat anti-rabbit igg antibody as quality control band antibody respectively
ML will indicate O-shaped rabbit-anti, A type or Asia I type antibodies against foot-and-mouth disease virus as detection band antibody and be diluted to 0.2~1mg/mL, point
Quality control band antibody and detection band antibody are not sprayed on nitrocellulose filter respectively by the discharge rate of 1~2 μ L/cm, form quality control band
With detection band, after 37 DEG C of dryings, it is placed in 3~5min of immersion, 37 DEG C of dryings after taking-up in confining liquid；
3) test card is assembled；
The restriction of the not no chronological order of the step 1) and step 2).
Preferably step 1) the colloidal gold buffer be containing mass percentage be 5~15% trehalose, quality
The pH value for the NP-40 that the casein and mass percentage that percentage composition is 1% are 0.5~1 ‰ is 7.4, molar concentration is
The phosphate buffer of 0.11mol/L.
Preferably, the step 2) confining liquid be containing mass percentage be 2.5~5% bovine serum albumin(BSA) and matter
Measuring the pH value that percentage composition is 0.5~1 ‰ NP-40 is 7.4, and molar concentration is the phosphate buffer of 0.11mol/L.
The present invention provides foot and mouth disease virus quantitative testing test paper cards and preparation method thereof.Test card of the present invention can
O-shaped aftosa in tissue or fluid sample, A type or Asia I type virus and its content are determined, meanwhile, this test card counterpart hoof
The detection of antigenic content in epidemic disease inactivated vaccine semi-finished product, finished product, can monitor in process of production and control the quality of vaccine；It is right
For vaccine user, test card screening high-quality vaccine can be used, inoculation failure is avoided.The inspection of existing foot and mouth disease virus content
The longer or operating method of required time is complex mostly for survey method, needs simple and quick detection side in practical applications
Method, this test paper test card is easy to operate quickly, and cost is relatively low, is suitable for field sample detection and production application.
Detailed description of the invention
Fig. 1 is test card structural schematic diagram provided by the invention；
Fig. 2 is foot and mouth disease virus quantitative testing test paper card testing result figure provided by the invention；
Fig. 3 is the O-shaped Virus Standard curve of aftosa；
Fig. 4 is foot and mouth disease A-type virus standard curve；
Fig. 5 is aftosa Asia I type Virus Standard curve.
Specific embodiment
The present invention provides foot and mouth disease virus quantitative testing test paper cards.In the present invention, the sample of the test card detection
Including aftosa vaccine semi-finished product, aftosa vaccine finished product, the tissue containing foot and mouth disease virus or containing the liquid-like of foot and mouth disease virus
Product.
In the present invention, the aftosa vaccine finished product carries out demulsification processing before detection；It is described containing foot and mouth disease virus
Tissue carries out decentralized processing before detection；The fluid sample containing foot and mouth disease virus is centrifuged before detection, takes supernatant.?
In the present invention, vaccine semi-finished product do not need to handle.
In the present invention, demulsification processing are as follows: by aftosa vaccine finished product and demulsifier according to mass ratio be (4~5):
1 mixing, 3000~5000rpm is centrifuged 10~15min after oscillation, takes lower liquid；
The demulsifier is n-butanol, n-amyl alcohol or trichloro ethylene.
In the present invention, the decentralized processing are as follows: the tissue containing foot and mouth disease virus is ground, is then pressed with dispersing agent
It is 1:5 mixing according to mass ratio, overnight, next day is centrifuged 5~10min with 3000~5000rpm to 4 DEG C of leaching poison, takes supernatant；
The dispersing agent is 0.11mol/L phosphate buffer or DMEM culture solution.
The present invention also provides the preparation methods of foot and mouth disease virus quantitative testing test paper card described in above-mentioned technical proposal, including
Following steps:
1) pH to 8.0~8.5 for adjusting colloidal gold solution, adds final concentration of 2~15 μ g/ml O-shaped, A type or Asia I type
Antibodies against foot-and-mouth disease virus, stir 30~40min, be added final concentration of 0.5~1% casein, stir 30~40min, 8000
~10000rpm is centrifuged 30~60min, discards supernatant, and gained precipitating is redissolved in the colloid of colloidal gold solution volume 1/10~1/20
In golden buffer, immune colloid gold solution is obtained, the immune colloid gold solution is sprayed, obtains indicating that rabbit-anti is O-shaped, A
The gold-labelled pad of type or Asia I type foot-and-mouth disease antiviral antibody；
2) phosphate buffer is used, is diluted to 0.2~1mg/ for goat anti-rabbit igg antibody as quality control band antibody respectively
ML will indicate O-shaped rabbit-anti, A type or Asia I type antibodies against foot-and-mouth disease virus as detection band antibody and be diluted to 0.2~1mg/mL, point
Quality control band antibody and detection band antibody are not sprayed on nitrocellulose filter respectively by the discharge rate of 1~2 μ L/cm, form quality control band
With detection band, after 37 DEG C of dryings, it is placed in 3~5min of immersion, 37 DEG C of dryings after taking-up in confining liquid；
3) test card is assembled；
The restriction of the not no chronological order of the step 1) and step 2).
The present invention adjusts the pH to 8.0~8.5 of colloidal gold solution, adds final concentration of 2~15 μ g/ml O-shaped, A type or Asia
I type antibodies against foot-and-mouth disease virus stirs 30~40min, and final concentration of 0.5~1% casein is added, and stirs 30~40min,
8000~10000rpm is centrifuged 30~60min, discards supernatant, and gained precipitating is redissolved in colloidal gold solution volume 1/10~1/20
In colloidal gold buffer, immune colloid gold solution is obtained, the immune colloid gold solution is sprayed, obtains indicating rabbit-anti O
The gold-labelled pad of type, A type or Asia I type foot-and-mouth disease antiviral antibody.In the present invention, it is preferred to the K of 0.2mol/L2CO3Adjust colloid
The pH value of gold solution.Immune colloid gold solution of the present invention is preferably kept under 4 DEG C of environment before spraying.In the present invention,
The colloidal gold solution is preferably prepared by trisodium citrate reduction method, and specifically, the preparation method is preferred are as follows: takes 0.01%
Aqueous solution of chloraurate 1000mL is heated to boiling, while agitation while 1% citric acid three sodium solution 15mL is added, to solution by black by
Gradually switch to red, continue to boil 5min, stops heating and being cooled to room temperature.The colloidal gold solution prepared is under spectrophotometer
Detecting its maximum absorption peak value is 523nm.In the present invention, it is 5 that the colloidal gold buffer, which preferably contains mass percentage,
The pH value for the NP-40 that the casein and mass percentage that~15% trehalose, mass percentage are 1% are 0.5~1 ‰
The phosphate buffer for being 0.11mol/L for 7.4, molar concentration.In the present invention, the immune colloid gold solution is sprayed
The method of painting is preferred are as follows: according to the discharge rate of 10-15 μ L/cm, is sprayed on all-glass paper with three-dimensional planar point spray instrument, 37 DEG C dry
It is sealed after dry.
The present invention uses phosphate buffer, 0.2 is diluted to using goat anti-rabbit igg antibody as quality control band antibody respectively~
1mg/mL will indicate O-shaped rabbit-anti, A type or Asia I type antibodies against foot-and-mouth disease virus as detection band antibody and be diluted to 0.2~1mg/
Quality control band antibody and detection band antibody are sprayed on nitrocellulose filter by the discharge rate of 1~2 μ L/cm respectively respectively, form matter by mL
Band and detection band are controlled, after 37 DEG C of dryings, is placed in 3~5min of immersion, 37 DEG C of dryings after taking-up in confining liquid.Quality Control of the present invention
Band and detection band are preferably by the distance of 5mm.In the present invention, it is 2.5~5% that the confining liquid, which is containing mass percentage,
Bovine serum albumin(BSA) and mass percentage are that the pH value of 0.5~1 ‰ NP-40 is 7.4, and molar concentration is the phosphorus of 0.11mol/L
Phthalate buffer.
The present invention assembles test card.It is also preferable to include the plastic clips of outer layer for test card of the present invention.The present invention
The structure of the papery card is that middle section of the PVC liner plate on bottommost, liner plate has nitrocellulose filter, nitrocellulose filter top
Absorption pad is posted in left end, and nitrocellulose filter upper right end is equipped with gold-labelled pad, and gold-labelled pad right end is equipped with sample pad.The suction of test card
The joining place for receiving pad and gold-labelled pad and nitrocellulose filter has overlapping, also has between gold-labelled pad and sample pad overlapping.By above-mentioned examination
Paper card is fitted into plastic clip, and the position corresponding to sample pad is equipped with well, and the position corresponding to nitrocellulose filter, which is equipped with, to be seen
Survey window.Assemble method of the present invention is preferred are as follows: the nitrocellulose filter containing quality control band and detection band is sticked on PVC liner plate,
Colloidal gold pad, sample pad are sticked in detection band lower section, absorption pad is sticked above quality control band, the PVC board that then will entirely glue
It is fitted into plastic clip, the position corresponding to sample pad is equipped with well, and the position corresponding to nitrocellulose filter is equipped with observation window.
Test card of the present invention is able to detect that aftosa vaccine semi-finished product, finished product, other are containing foot and mouth disease virus tissue or the samples such as liquid
The viral level of product.
In the present invention, the application method of the test card are as follows: foot and mouth disease virus is O-shaped, A type or Asia I type virus mark
Quasi- product are added in test card, are put into gold strip reader after reaction 15min and are detected, it is known that the sample detection of various concentration
Obtained T/C value goes out standard curve with Function Fitting, and the viral level in sample to be tested can be calculated according to functional relation.Institute
State functional relation are as follows: y=ax+b, wherein y represents viral level (unit: μ g/ml), and x represents T/C value, and a represents slope, b generation
Table constant.Examination is added after aftosa of the present invention is O-shaped, A type or Asia I type Virus Standard product preferably carry out 2 times of gradient dilutions
In paper card, additional amount is preferably 70 μ L.In the present invention, the model of the gold strip reader is preferably TSR-100A.This hair
It is bright that when quality control band does not occur red stripes, to be judged to result invalid；When red stripes occurs in quality control band, to be judged to result effective.By T/C value
It is brought into the viral level calculated in vaccine in corresponding calibration curve formula.
Combined with specific embodiments below to foot and mouth disease virus quantitative testing test paper card of the present invention and preparation method thereof
It is further described in detail, technical solution of the present invention includes but is not limited to following embodiment.
Embodiment 1
The preparation of the O-shaped Viral Quantification Test paper card of aftosa:
1. the preparation of colloidal gold: preparing colloidal gold using trisodium citrate reduction method, take 0.01% aqueous solution of chloraurate
1000ml is heated to boiling, and 1% citric acid three sodium solution 15ml is added in agitation, gradually switchs to red by black to solution,
Continue to boil 5min, stops heating and being cooled to room temperature.The colloidal gold solution prepared detects its highest under spectrophotometer
Absorption peak is 523nm.
2. the preparation (the O-shaped antibodies against foot-and-mouth disease virus of colloid gold label) of immune colloid gold
With the K of 0.2mol/L2CO3Colloidal gold solution is adjusted to pH8.0, the O-shaped hoof-and-mouth disease of final concentration of 2 μ g/ml is added
Malicious antibody stirs 30min, and final concentration of 1% casein is added in colloidal gold solution later, stirs 30min, 8000rpm
It is centrifuged 40min, is discarded supernatant, gained precipitating is redissolved in the colloidal gold buffer of colloidal gold solution volume 1/10, and 4 DEG C of guarantors are placed in
It deposits.
Above-mentioned colloidal gold buffer be containing 5% trehalose, 1% casein, 0.5 ‰ NP-40 pH7.4,0.11mol/L's
Phosphate buffer.
3. metal spraying
Respectively by the immune colloid gold prepared according to the discharge rate of 15 μ L/cm, with three-dimensional planar point spray instrument in glass
It sprays in fibrous paper, is sealed after 37 DEG C of dryings.
4. the preparation of quality control band and detection band on nitrocellulose filter
With pH7.4, the phosphate buffer of 0.02mol/L is diluted goat anti-rabbit igg antibody as quality control band antibody respectively
To 0.5mg/mL, the work for being diluted to 2mg/mL with antibody using O-shaped, A type or Asia I type foot-and-mouth disease virus rabbit-anti as detection is dense
Both the above antibody, is sprayed on nitrocellulose filter by the discharge rate of 2 μ L/cm by degree respectively respectively, forms quality control band and detection band,
It at a distance of 5mm between two bands, after 37 DEG C dry, be placed in confining liquid and impregnate 5min, 37 DEG C of dryings and be sealed after taking-up.
Above-mentioned confining liquid is the bovine serum albumin(BSA) containing 2.5%, the phosphate of the pH7.4 of 0.5 ‰ NP-40,0.11mol/L
Buffer.
5. the assembling of test card
The nitrocellulose filter containing quality control band and detection band is sticked on PVC liner plate, sticks colloidal gold in detection band lower section
Pad, sample pad, absorption pad is sticked above quality control band, then the PVC board entirely glued is fitted into plastic clip, corresponds to sample
The position of pad is equipped with well, and the position corresponding to nitrocellulose filter is equipped with observation window.
6. the fitting of standard curve
The O-shaped Virus Standard product of foot and mouth disease virus are subjected to 2 times of gradient dilutions, respectively by the sample drop of the 70 each dilutions of μ L
Enter well, after reacting 15min, is placed in gold strip reader and is detected (see Fig. 2), read T/C value (Parallel testing 3
It is secondary), as shown in table 1.
1 T/C value result of table
Standard curve (as shown in Figure 3) is fitted according to the concentration of T/C value and respective standard product, generates the letter of standard curve
Number relational expression.
The processing of vaccine sample:
The O-shaped inactivated vaccine of 1mL aftosa and demulsifier are mixed in 4:1 ratio, fullyd shake, 3000rpm centrifugation
10min, draws lower layer's antigen liquid, and 4 DEG C of preservations are to be checked.
The detection of vaccine sample:
70 μ L of the sample handled well is taken to be added drop-wise in the O-shaped Viral Quantification Test paper card of aftosa, after reacting 15min,
Test strips are placed in gold strip reader, T/C value is read.
Result judgement:
When quality control band does not occur red stripes, to be judged to result invalid；When red stripes occurs in quality control band, to be judged to result effective.
It T/C value is brought into corresponding calibration curve formula y=4.1759x-1.0134 calculates the virus in vaccine and contain
It measures (μ g/mL).
Embodiment 2
The preparation of foot and mouth disease A-type virus quantitative testing test paper card:
1. the preparation of colloidal gold: preparing colloidal gold using trisodium citrate reduction method, take 0.01% aqueous solution of chloraurate
1000ml is heated to boiling, and 1% citric acid three sodium solution 15ml is added in agitation, gradually switchs to red by black to solution,
Continue to boil 5min, stops heating and being cooled to room temperature.The colloidal gold solution prepared detects its highest under spectrophotometer
Absorption peak is 523nm.
2. the preparation (colloid gold label A type antibodies against foot-and-mouth disease virus) of immune colloid gold
With the K of 0.2mol/L2CO3Colloidal gold solution is adjusted to pH8.0, final concentration of 5 μ g/mlA type foot and mouth disease virus is added
Antibody stirs 30min, is added final concentration of 1% casein in colloidal gold solution later, stirs 30min, 8000rpm from
Heart 40min, discards supernatant, and gained precipitating is redissolved in the colloidal gold buffer of colloidal gold solution volume 1/15, is placed in 4 DEG C of guarantors
It deposits.
Above-mentioned colloidal gold buffer be containing 5% trehalose, 1% casein, 0.5 ‰ NP-40 pH7.4,0.11mol/L's
Phosphate buffer.
3. metal spraying
Respectively by the immune colloid gold prepared according to the discharge rate of 15 μ L/cm, with three-dimensional planar point spray instrument in glass
It sprays in fibrous paper, is sealed after 37 DEG C of dryings.
4. the preparation of quality control band and detection band on nitrocellulose filter
With pH7.4, the phosphate buffer of 0.02mol/L is diluted goat anti-rabbit igg antibody as quality control band antibody respectively
To 0.5mg/mL, the working concentration of 4mg/mL is diluted to antibody using A type foot and mouth disease virus rabbit-anti as detection, presses 2 μ L/ respectively
Both the above antibody is sprayed on nitrocellulose filter by the discharge rate of cm respectively, forms quality control band and detection band, phase between two bands
It away from 5mm, after 37 DEG C dry, be placed in confining liquid and impregnate 5min, 37 DEG C of dryings and be sealed after taking-up.
Above-mentioned confining liquid is the bovine serum albumin(BSA) containing 2.5%, the phosphate of the pH7.4 of 0.5 ‰ NP-40,0.11mol/L
Buffer.
5. the assembling of test card
The nitrocellulose filter containing quality control band and detection band is sticked on PVC liner plate, sticks colloidal gold in detection band lower section
Pad, sample pad, absorption pad is sticked above quality control band, then the PVC board entirely glued is fitted into plastic clip, corresponds to sample
The position of pad is equipped with well, and the position corresponding to nitrocellulose filter is equipped with observation window.
6. the fitting of standard curve
Foot and mouth disease virus A type Virus Standard product are subjected to 2 times of gradient dilutions, respectively by the sample drop of the 70 each dilutions of μ L
Enter well, after reacting 15min, be placed in gold strip reader and detected, reads T/C value, the results are shown in Table 2.
2 T/C value result of table
Standard curve (as shown in Figure 4) is fitted according to the concentration of T/C value and respective standard product, generates the letter of standard curve
Number relational expression.
Processing containing foot and mouth disease virus tissue or the fluid sample containing foot and mouth disease virus:
Animal body tissue is fully ground according to being placed in tissue refiner, the group that 1:5 is made in tissue dispersion agent is added
Suspension is knitted, overnight, next day is centrifuged 5min with 10000rpm to 4 DEG C of leaching poison, takes supernatant spare.
Blister fluid is centrifuged without milled processed with 3000rpm, takes supernatant spare.
The detection of sample to be tested:
It takes 70 μ L of the sample handled well to be added drop-wise in A type test card well, after reacting 15min, test strips is placed in gold
It marks in a reading apparatus, reads T/C value.
Result judgement
When quality control band does not occur red stripes, to be judged to result invalid；When red stripes occurs in quality control band, to be judged to result effective.
T/C value is brought into corresponding calibration curve formula y=5.4254x-1.073 and calculates viral level in vaccine
(μg/mL)。
Embodiment 3
The preparation of aftosa Asia I type Viral Quantification Test paper card:
1. the preparation of colloidal gold: preparing colloidal gold using trisodium citrate reduction method, take 0.01% aqueous solution of chloraurate
1000ml is heated to boiling, and 1% citric acid three sodium solution 15ml is added in agitation, gradually switchs to red by black to solution,
Continue to boil 5min, stops heating and being cooled to room temperature.The colloidal gold solution prepared detects its highest under spectrophotometer
Absorption peak is 523nm.
2. the preparation (colloid gold label Asia I type antibodies against foot-and-mouth disease virus) of immune colloid gold
With the K of 0.2mol/L2CO3Colloidal gold solution is adjusted to pH8.0, final concentration of 1 μ g/ml Asia I type aftosa is added
Antiviral antibody stirs 30min, and final concentration of 1% casein is added in colloidal gold solution later, stirs 30min,
8000rpm is centrifuged 40min, discards supernatant, and gained precipitating is redissolved in the colloidal gold buffer of colloidal gold solution volume 1/10, sets
It is saved in 4 DEG C.
Above-mentioned colloidal gold buffer be containing 5% trehalose, 1% casein, 0.5 ‰ NP-40 pH7.4,0.11mol/L's
Phosphate buffer.
3. metal spraying
Respectively by the immune colloid gold prepared according to the discharge rate of 15 μ L/cm, with three-dimensional planar point spray instrument in glass
It sprays in fibrous paper, is sealed after 37 DEG C of dryings.
4. the preparation of quality control band and detection band on nitrocellulose filter
With pH7.4, the phosphate buffer of 0.02mol/L is diluted goat anti-rabbit igg antibody as quality control band antibody respectively
To 0.5mg/mL, the working concentration of 4mg/mL is diluted to antibody using Asia I type foot and mouth disease virus rabbit-anti as detection, is pressed respectively
Both the above antibody is sprayed on nitrocellulose filter by the discharge rate of 2 μ L/cm respectively, formed quality control band and detection band, two bands it
Between at a distance of 5mm, after 37 DEG C are dry, are placed in confining liquid and impregnate 5min, 37 DEG C of dryings and be sealed after taking-up.
Above-mentioned confining liquid is the bovine serum albumin(BSA) containing 2.5%, the phosphate of the pH7.4 of 0.5 ‰ NP-40,0.11mol/L
Buffer.
5. the assembling of test card
The nitrocellulose filter containing quality control band and detection band is sticked on PVC liner plate, sticks colloidal gold in detection band lower section
Pad, sample pad, absorption pad is sticked above quality control band, then the PVC board entirely glued is fitted into plastic clip, corresponds to sample
The position of pad is equipped with well, and the position corresponding to nitrocellulose filter is equipped with observation window.
6. the fitting of standard curve
Foot and mouth disease virus Asia I type Virus Standard product are subjected to 2 times of gradient dilutions, respectively by the sample of the 70 each dilutions of μ L
Product instill well, after reacting 15min, are placed in gold strip reader and are detected, and read T/C value, as shown in table 3.
3 T/C value result of table
Standard curve (as shown in Figure 5) is fitted according to the concentration of T/C value and respective standard product, generates the letter of standard curve
Number relational expression.
The processing of inactivated foot-and-mouth disease vaccine semi-finished product:
Inactivated vaccine before not emulsified can be detected directly as measuring samples.
The detection of sample to be tested:
It takes 70 μ L of sample to be tested to be added drop-wise in Asia I type test card well, after reacting 15min, test strips is placed in gold
It marks in a reading apparatus, reads T/C value.
Result judgement
When quality control band does not occur red stripes, to be judged to result invalid；When red stripes occurs in quality control band, to be judged to result effective.
T/C value is brought into corresponding calibration curve formula y=5.554x-0.1572 and calculates viral level in vaccine
(μg/mL)。
Conventional aftosa Test paper card is as just the yin and yang attribute of qualitative detection sample, and the present invention is in addition to can be qualitative
It detects outside aftosa sample, it is often more important that quantitative detection can be carried out to positive sample, it is so specific that determine virus in sample
Content can be applied in vaccine finished product or semi-finished product detection deeper into ground.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (8)
1. foot and mouth disease virus quantitative testing test paper card, the test card include get stuck (1), PVC liner plate (2), absorption pad (3), nitre
Acid cellulose film (4), gold-labelled pad (5) and sample pad (6), which is characterized in that O-shaped rabbit-anti, A type or Asia are indicated in the gold-labelled pad
Continent I type antibodies against foot-and-mouth disease virus；Coating indicates the quality control band (7) of goat anti-rabbit igg antibody and indicates on the nitrocellulose filter
Rabbit-anti is O-shaped, the detection band (8) of A type or Asia I type antibodies against foot-and-mouth disease virus.
2. test card according to claim 1, which is characterized in that the sample of the test card detection includes aftosa vaccine
Semi-finished product, aftosa vaccine finished product, the tissue containing foot and mouth disease virus or the fluid sample containing foot and mouth disease virus.
3. test card according to claim 2, which is characterized in that the aftosa vaccine finished product is demulsified before detection
Processing；The tissue containing foot and mouth disease virus carries out decentralized processing before detection；The fluid sample containing foot and mouth disease virus exists
It is centrifuged before detection, takes supernatant.
4. test card according to claim 3, which is characterized in that demulsification processing are as follows: by aftosa vaccine finished product with
Demulsifier is (4~5) according to mass ratio: 1 mixing, 3000~5000rpm is centrifuged 10~15min after oscillation, takes lower liquid；
The demulsifier is n-butanol, n-amyl alcohol or trichloro ethylene.
5. test card according to claim 3, which is characterized in that the decentralized processing are as follows: by the group containing foot and mouth disease virus
Knit and ground, be then that 1:5 mix according to mass ratio with dispersing agent, 4 DEG C of leachings are malicious overnight, next day with 3000~5000rpm from
5~10min of the heart, takes supernatant；
The dispersing agent is 0.11mol/L phosphate buffer or DMEM culture solution.
6. the preparation method of any one of Claims 1 to 5 foot and mouth disease virus quantitative testing test paper card, comprising the following steps:
1) pH to 8.0~8.5 for adjusting colloidal gold solution, adds final concentration of 2~15 μ g/ml O-shaped, A type or Asia I type mouth hoof
Epidemic disease antiviral antibody, stir 30~40min, be added final concentration of 0.5~1% casein, stir 30~40min, 8000~
10000rpm is centrifuged 30~60min, discards supernatant, and gained precipitating is redissolved in the colloidal gold of colloidal gold solution volume 1/10~1/20
In buffer, immune colloid gold solution is obtained, the immune colloid gold solution is sprayed, obtains indicating that rabbit-anti is O-shaped, A type
Or the gold-labelled pad of Asia I type foot-and-mouth disease antiviral antibody；
2) phosphate buffer is used, is diluted to 0.2~1mg/mL for goat anti-rabbit igg antibody as quality control band antibody respectively, it will
O-shaped rabbit-anti, A type or Asia I type antibodies against foot-and-mouth disease virus are indicated as detection band antibody and is diluted to 0.2~1mg/mL, presses 1 respectively
Quality control band antibody and detection band antibody are sprayed on nitrocellulose filter by the discharge rate of~2 μ L/cm respectively, form quality control band and detection
Band after 37 DEG C of dryings, is placed in 3~5min of immersion, 37 DEG C of dryings after taking-up in confining liquid；
3) test card is assembled；
The restriction of the not no chronological order of the step 1) and step 2).
7. preparation method according to claim 6, which is characterized in that step 1) the colloidal gold buffer is containing quality hundred
The casein and mass percentage that trehalose that point content is 5~15%, mass percentage are 1% are 0.5~1 ‰
The phosphate buffer that the pH value of NP-40 is 7.4, molar concentration is 0.11mol/L.
8. preparation method according to claim 6, which is characterized in that the step 2) confining liquid is containing mass percentage
Bovine serum albumin(BSA) and mass percentage for 2.5~5% are that the pH value of 0.5~1 ‰ NP-40 is 7.4, and molar concentration is
The phosphate buffer of 0.11mol/L.
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Application publication date: 20191001 |