CN105954514A - Bigeminal rapid detection test strip for foot-and-mouth disease and pseudorabies and preparation method thereof - Google Patents
Bigeminal rapid detection test strip for foot-and-mouth disease and pseudorabies and preparation method thereof Download PDFInfo
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- CN105954514A CN105954514A CN201610465660.6A CN201610465660A CN105954514A CN 105954514 A CN105954514 A CN 105954514A CN 201610465660 A CN201610465660 A CN 201610465660A CN 105954514 A CN105954514 A CN 105954514A
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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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- 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
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
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Abstract
The invention relates to a bigeminal rapid detection test strip for foot-and-mouth disease and pseudorabies and a preparation method thereof. Based on a film Immunochromatography principle, the test strip is prepared by adopting a colloidal gold double-antigen sandwich method; and staphylococcus protein A (SPA) is used as a capturing antigen and a foot-and-mouth disease virus 3ABC and a pseudorabies virus GE protein are used as detection antigens. When the test strip is used for detecting a blood serum sample, the test strip can be used for diagnosing wild virus infection of the foot-and-mouth disease and the pseudorabies of pigs at the same time. The test strip is convenient to operate and instruments and equipment are not needed; and a result is simple to judge and the requirements on diagnosis of important diseases by primary organizations and farmers can be met, so that the test strip has important meanings for prevention and control of the foot-and-mouth disease and the pseudorabies of the pigs.
Description
Technical field
This patent disclosure relates generally to animal epidemic quick diagnosis test strips, be specifically related to foot and mouth disease and puppet
Rabies bigeminy Rapid detection test strip.
Background technology
Foot and mouth disease (foot-and-mouth disease, FMD) is a kind of acute, hot, high
Degree contagious disease, the artiodactyls such as pig, cattle, sheep is the most susceptible.The morbidity of Schweineseuche
Rate is high, propagates rapidly, can cause the large quantities of death of Sow abortion, piglet, bring serious economy
Loss.Foot and mouth disease is taked compulsory immunization strategy by China, the most all can use substantial amounts of inactivation epidemic disease
Seedling, and monitor immune effect of vaccine, improve animal clinical antibody horizontal.How to distinguish vaccine to exempt from
Epidemic disease animal and wild virus infection animal become the key affecting foot and mouth disease prevention and control.Raw at foot-and-mouth disease vaccine
During product, 3ABC non-structural protein can be removed, and therefore animal inoculation foot-and-mouth disease vaccine is not
Produce 3ABC antibody.Therefore, detect 3ABC protein antibodies, can be as diagnosis wild poison sense
The important indicator of dye.
Pseudorabies (Pseudorabies virus, PRV) is the acute infectious disease of a boar,
Mainly encroach on reproductive system, may result in sow infertile, miscarriage, stillborn fetus, suckling pig is the most dead
Die, adult growing and fattening pigs pig can cause growth retardation, weightening finish slowly, be harm pig industry
One of serious infectious diseases.Vaccine virus immunization is the important measures of prevention and control pseudorabies.At present,
Can available pseudorabies vaccine be attenuated live vaccines, differentiate that immune animal and wild virus infection move
Thing is the key that pseudorabies purifies.Disappearance GE protein gene is built by technique for gene engineering
Pseudorabies virus mutant, prepared vaccinated animals, will not induce generation GE egg
Bai Kangti.So, by detection GE protein antibodies, pseudorabies vaccine can be accurately distinguished and exempt from
Epidemic disease swinery and wild virus infection swinery.
Along with the development of biotechnology, there are Schweineseuche 3ABC and puppet
Rabies GE antibody assay kit, but mainly elisa (ELISA)
Detection method.ELISA complex operation, the longest, and need instrument, it is unfavorable for clinic
Medical diagnosis on disease on the spot.Occur in that the colloid of a kind of similar patent CN103792373A afterwards
Gold rapid detection card, but can only single diagnosis pseudorabies wild virus infection.Schweineseuche, puppet
Farrowing sow and piglet are endangered big by rabies, and heavy damage pig industry sustainable development is developed
Quickly, multi-joint medical diagnosis on disease technology is significant.
Summary of the invention
It is an object of the invention to: a kind of animal epidemic quick diagnosis test strips and preparation thereof are provided
Method, this test strips can diagnose the wild virus infection of Schweineseuche and pseudorabies simultaneously.
The present invention is achieved through the following technical solutions: this test strips includes base plate, coated film, glass
Glass fibrous membrane, absorbent paper and sample pad；Wherein, the bottom surface of coated film is pasted onto on base plate, bag
The two ends in tunicle front sticking glass fibrous membrane and absorbent paper respectively；At glass fibre membrane away from suction
Sample pad is pasted in the front of water paper one end；Coated film is provided with detection line I, detection line II
And nature controlling line, detection line I is coated FMDV 3ABC, and detection line II is coated puppet
Rabies virus GE albumen, nature controlling line is coated rabbit staphylococcus A protein I gG antibody；Glass
Staphylococcal protein A (SPA) colloid gold particle production it is coated with on glass fibrous membrane.
Test strips application dual-antigen sandwich method of the present invention, when in test sample containing 3ABC or
During GE antibody, then at sample pad with SPA protein binding, form that " SPA Ag-Ab is multiple
Compound ", and under the effect of absorbent paper, permeate forward swimming.When complex arrives detection line I,
3ABC antibody occurs specific binding with FMDV 3ABC, and " SPA resists in formation
Former-3ABC antibody-3ABC antigenic compound ", coated film occurs macroscopic color
Band.When complex arrives detection line II, and GE antibody occurs spy with Pseudorabies virus GE albumen
Anisogamy, formed " SPA antigen-GE antibody-GE antigenic compound ", on coated film
Macroscopic colour band occurs.When in test sample without 3ABC and GE antibody, colloid
The SPA swimming of gold labelling to nature controlling line, and rabbit staphylococcus A protein I gG antibodies,
Coated film occurs macroscopic colour band.During detection, draw blood serum sample to be checked 1,
Drip in sample pad, observe detection line I, II and whether nature controlling line red ribbon occurs, thus
Judge that whether animal is by foot and mouth disease, pseudorabies wild virus infection.
The positive effect of the present invention is:
1, simple operation of the present invention, it is not necessary to instrument and equipment, result interpretation is simple.In detection
During a blood serum sample, Schweineseuche and pseudorabies wild virus infection can be diagnosed simultaneously.Can expire
Foot grassroots organization, peasant household are for the demand of great disease diagnosis.
2, the present invention uses dual-antigen sandwich method, is not limited by the Hosts of sample, except
Diagnosis Schweineseuche and pseudorabies wild virus infection, it may also be used for the poison sense of many animals foot and mouth disease open country
The Differential Diagnosis of dye.
3, immunochromatographic method is combined by the present invention with colloidal gold technique, develops quick diagnosis
Test strips.Relative to ELISA, test strips supplementary material is the simplest, and low cost is of value to
Popularization and application.
Accompanying drawing explanation
Fig. 1 is the side structure schematic diagram of test strips of the present invention.In figure 1: base plate；2: bag
Tunicle；3: glass fibre membrane；4: absorbent paper；5: sample pad.
Fig. 2 is the Facad structure schematic diagram of Fig. 1.In figure 6: detection line I；7: detection line II；
8: nature controlling line.
Fig. 3 is the foot and mouth disease wild virus infection positive and pseudorabies wild virus infection positive findings schematic diagram.
All there is red ribbon in detection line I6, detection line II7 and nature controlling line 8 on coated film 2.
Fig. 4 is the foot and mouth disease wild virus infection positive and pseudorabies wild virus infection negative findings schematic diagram.
All there is red ribbon in detection line I6 and nature controlling line 8 on coated film 2, and detection line II7 is not
Red ribbon occurs.
Fig. 5 is the pseudorabies wild virus infection positive and foot and mouth disease wild virus infection negative findings schematic diagram.
All there is red ribbon in detection line II7 and nature controlling line 8 on coated film 2, and detection line I6 is not
Red ribbon occurs.
Fig. 6 is foot and mouth disease wild virus infection feminine gender and pseudorabies wild virus infection negative findings schematic diagram.
There is red ribbon, detection line I6 and detection line II7 the most not in nature controlling line 8 on coated film 2
Red ribbon occurs.
Fig. 7 is that the present invention detects null result schematic diagram.Nature controlling line 8 on coated film 2, inspection
Survey line I6 and detection line II7 all occurs without red ribbon.
Fig. 8 is that the present invention detects null result schematic diagram.Nature controlling line 8 on coated film 2 does not goes out
There is red ribbon in existing red ribbon, detection line I6 and detection line II7.
Fig. 9 is that the present invention detects null result schematic diagram.Nature controlling line 8 on coated film 2 and inspection
Survey line I6 all occurs without red ribbon, and red ribbon occurs in detection line II7.
Figure 10 is that the present invention detects null result schematic diagram.Nature controlling line 8 He on coated film 2
Detection line II7 all occurs without red ribbon, and red ribbon occurs in detection line I6.
Detailed description of the invention
Foot and mouth disease and the pseudorabies bigeminal of the most specifically introducing the present invention are quickly examined
Test paper slip and preparation method thereof.It should be appreciated by those skilled in the art that enforcement described below
Example is intended merely to be more fully understood that the present invention, is not intended to limit the present invention.
As it is shown in figure 1, according to the foot and mouth disease of the present invention and pseudorabies bigeminal quick detection test paper
Bar includes base plate 1, coated film 2, glass fibre membrane 3, absorbent paper 4 and sample pad 5.Its
In, coated film 2 is pasted onto above base plate 1, the two ends in coated film 2 front sticking glass respectively
Fibrous membrane 3 and absorbent paper 4.Glue away from the front of one end of absorbent paper 4 at glass fibre membrane 3
Patch sample pad 5.Base plate 1 usually PVC material, coated film 2 is nitrocellulose filter (NC
Film).Glass fibre membrane 3 is coated with colloid gold label SPA albumen.
As in figure 2 it is shown, be provided with detection line I6, detection line II7 and Quality Control on coated film 2
Line 8.Detection line I6 is coated FMDV 3ABC, and detection line II7 is coated pseudo-mad
Dog disease poison GE albumen, nature controlling line 8 is coated rabbit staphylococcus A protein I gG antibody.Institute
State detection line I6, detection line II7 and nature controlling line 8 on coated film 2 in 3 line parallel
According to detection line I6, detection line II7 and nature controlling line 8, whether row, occur that red ribbon enters
Row result judges.
Main raw material(s) source in following embodiment is: FMDV 3ABC,
Pseudorabies virus GE albumen is prepared by Zhengzhou Zhong Dao Bioisystech Co., Ltd.Other reagent and
Material all can derive from commercial sources.Described experimental technique, unless otherwise noted, is often
Rule experimental technique.
Test strips of the present invention uses the colloidal gold immunity chromatography of 3 band colour developings to prepare, aobvious
Showing that band is red ribbon, concrete preparation method is as follows:
The preparation of 1.1 coated films 2
Nitrocellulose filter sprays FMDV 3ABC, pseudorabies respectively
Poison GE albumen and rabbit staphylococcus A protein I gG antibody, and arranged in parallel in 3 lines,
Packet composition detection line I6, detection line II7 and nature controlling line 8.37 DEG C of drying and processings 2 hours,
Put and save backup equipped with in the sealing bag of desiccant.
Detection line I6: debugging spray film instrument, spouting liquid is 1ul/cm, with being coated buffer dilution
FMDV 3ABC, concentration is 2.5mg/mL, with spray film instrument spraying line.
Detection line II7: debugging spray film instrument, spouting liquid is 1ul/cm, with being coated buffer dilution
Pseudorabies virus GE albumen, concentration is 3.0mg/mL, with spray film instrument spraying line.
Nature controlling line 8: debugging spray film instrument, spouting liquid is 1ul/cm, with being coated buffer dilution rabbit
Staphylococcus A protein I gG antibody, concentration is 1mg/mL, with spray film instrument spraying line.
The described buffer formulation that is coated: weigh bovine serum albumin 0.01g, 12 hypophosphite monohydrates
Disodium hydrogen 30.072g, potassium dihydrogen phosphate 2.176g, dissolve with ultra-pure water, and be settled to
1000mL。
Being drawn 3 lines on coated film 2 and answer fine uniform, adjacent line-to-line is every 0.4-1.0cm.
The preparation of 1.2 glass fibre membranes 3
(1) preparation of colloidal gold solution
Being placed on magnetic stirring apparatus by distilled water and be heated to boiling, being rapidly added concentration is 1%
Chlorauric acid solution is to final concentration of 0.02%.Boiling 5 minutes, adding concentration afterwards is 1%
Citric acid three sodium solution, injection volume is 1.8 times of chlorauric acid solution.After boiling 10 minutes,
Being cooled to room temperature, then be 0.02% by distilled water adjustment gold chloride concentration, room temperature keeps in Dark Place standby
With.
(2) preparation of colloid gold label SPA glass fibre membrane 3
With 5M potassium carbonate regulation colloidal gold solution pH value to 7.5-8.0, by 60ug antibody/mL
The ratio of colloidal gold solution adds SPA albumen, stands 5 minutes after mixing.Again by 5% body
Long-pending amount adds 10% bovine serum albumin solution, stands 5 minutes after mixing.10000rpm
Centrifugal 30 minutes, supernatant discarded, wash 1 time with labelling cleaning mixture.Precipitation is with 1/10th
The gold mark albumen of initial colloid gold volume preserves liquid and dissolves, then according to every milliliter of solution paving
10cm2Amount be sprayed on glass fibre membrane 3.It is vacuum dried 3 hours, puts equipped with desiccant
Sealing bag in save backup.
Described 10% bovine serum albumin solution: take bovine serum albumin 100g, use distilled water
It is settled to 1000mL.
Described labelling cleaning mixture: take bovine serum albumin 4g, PEG 8000 2g, poly-second
Alkene pyrrolidone 2g, sucrose 2g, Tris 1.211g, be settled to 1000mL with distilled water, and
Regulation PH to 8.1.
Described gold mark albumen preserves liquid: take bovine serum albumin 25g, PEG 8000 5g,
Tris 10.2g, sodium hydroxide 2.5g, polysorbas20 7.5mL, be settled to 1000mL with distilled water,
And regulate PH to 8.5.
1.3 big plate group bars
Each component specification (long × wide): base plate 1 (30cm × 7.6cm), coated film 2
(30cm × 2.0cm), sample pad 5 (30cm × 3.0cm), coating colloid gold label SPA egg
White glass fibre membrane 3 (30cm × 0.7cm) and absorbent paper 4 (30cm × 3.2cm).
The compound mode of each layer is as follows: be pasted onto by coated film 2 above base plate 1, coated film 2
The two ends in front sticking glass fibrous membrane 3 and absorbent paper 4 respectively.Glass fibre membrane 3 away from
Sample pad 5 is pasted in the front of one end of absorbent paper 4.I.e. paste the most successively on base plate 1
Coated film 2, glass fibre membrane 3, absorbent paper 4, sample pad 5.
1.4 cutting
With cutting cutter, big plate being cut into wall scroll, every width is 3-5cm.
The ELISA test strip principle and the result that describe the present invention the most again in detail judge.
2.1 ELISA test strip principles
Test strips application dual-antigen sandwich method of the present invention, with SPA albumen as capture antigen,
With foot and mouth disease virus 3ABC and Pseudorabies virus GE albumen as detection antigen.Its principle is
When in test sample containing 3ABC or GE antibody time, then at sample pad 5 with SPA egg
White combination, is formed " SPA antigen-antibody complex ", and under the effect of absorbent paper 4 forward
Infiltration swimming.When complex arrives detection line 16,3ABC antibody and foot and mouth disease virus 3ABC
Albumen occurs specific binding, and " SPA antigen-3ABC antibody-3ABC antigen is combined in formation
Thing ", coated film 2 occurs macroscopic colour band.When complex arrives detection line 117,
GE antibody occurs specific binding with Pseudorabies virus GE albumen, forms " SPA antigen-GE
Antibody-GE antigenic compound ", NC film occurs macroscopic colour band.When solution is swum
Move to nature controlling line 8, and rabbit staphylococcus A protein I gG antibodies, at coated film 2
The macroscopic colour band of upper appearance.During detection, draw blood serum sample to be checked 1, drip to sample
On pad 5, observe detection line I6, II7 and whether nature controlling line 8 red ribbon occurs, thus sentence
Whether determine animal by foot and mouth disease, pseudorabies wild virus infection.
2.2 test strips results judge
Below in conjunction with the accompanying drawings, the result criterion of test strips of the present invention is specifically introduced.
As it is shown on figure 3, detection line I6, detection line II7 and the nature controlling line 8 on coated film 2
Red ribbon all occur, then foot and mouth disease and pseudorabies are the wild virus infection positive.
As shown in Figure 4, all there is red color in the detection line I6 on coated film 2 and nature controlling line 8
Band, detection line II7 occurs without red ribbon, then foot and mouth disease wild virus infection is positive, and pseudorabies
Sick wild virus infection is negative.
As it is shown in figure 5, all there is red color in detection line II7 and nature controlling line 8 on coated film 2
Band, detection line I6 occurs without red ribbon, then the foot and mouth disease wild virus infection positive and pseudorabies
Wild virus infection is negative.
As shown in Figure 6, there is red ribbon in the nature controlling line 8 on coated film 2, detects line I6
All occur without red ribbon with detection line II7, then foot and mouth disease and pseudorabies are wild virus infection
Negative；
When several situation below occurring, testing result is invalid: nature controlling line 8 on coated film 2,
Detection line I6 and detection line II7 all occurs without red ribbon (Fig. 7)；Matter on coated film 2
Control line 8 occurs without red ribbon, detection line I6 and detection line II7 and red ribbon (figure occurs
8)；Nature controlling line 8 and detection line I6 on coated film 2 occur without red ribbon, detect line II
7 there is red ribbon (Fig. 9)；Nature controlling line 8 and detection line II7 on coated film 2 occur without
Red ribbon, there is red ribbon (Figure 10) in detection line I6.
Claims (3)
1. foot and mouth disease and a pseudorabies bigeminal Rapid detection test strip, this test strips includes
Base plate, coated film, glass fibre membrane, absorbent paper and sample pad；
Wherein, the bottom surface of coated film is pasted onto on base plate, and the two ends in coated film front are pasted respectively
Glass fibre membrane and absorbent paper；Glass fibre membrane away from absorbent paper one end front paste sample
Pad；Being provided with detection line I, detection line II and nature controlling line on coated film, detection line I is coated mouth
Aphtovirus 3ABC albumen, detection line II is coated Pseudorabies virus GE albumen, nature controlling line
It is coated rabbit staphylococcus A protein I gG antibody；Staphylococcus it is coated with on glass fibre membrane
A protein-colloid gold grain production.
Foot and mouth disease the most according to claim 1 and pseudorabies bigeminal quick detection test paper
Bar, wherein detection line I, detection line II and three lines of nature controlling line are in being arranged in parallel, and adjacent two
Line interval 0.4-1.0cm.
3. foot and mouth disease and a preparation method for pseudorabies bigeminal Rapid detection test strip, bag
Include following steps:
(1) by FMDV 3ABC, Pseudorabies virus GE albumen, Tu Kang Portugal
Grape coccus A protein I gG antibody respectively be coated buffer dilution after formed concentration be
The spray coating liquor of 1.0-3.0mg/mL；With the corresponding spray of spraying line respectively on coated film of spray film instrument
Masking liquid, forms detection line I, detection line II and nature controlling line；
(2) distilled water is placed on magnetic stirring apparatus be heated to boiling, be rapidly added gold chloride
Solution；Boiling and add citric acid three sodium solution afterwards, injection volume is the 1.5-3 of chlorauric acid solution
Times；After boiling 10-30 minute, it is cooled to room temperature and forms colloidal gold solution；
(3) with potassium carbonate regulation colloidal gold solution pH value to 7.5-8.0, by 60ug antibody/mL
The ratio of colloidal gold solution adds SPA albumen, stands after mixing；Again by the amount of 5% volume
Add 10% bovine serum albumin solution, stand after mixing；Centrifugal supernatant discarded, uses labelling
Cleaning mixture washs；The precipitate gold mark albumen of 1/10th initial colloid gold solution volumes preserves
Liquid dissolves, then according to every milliliter of solution paving 10-15cm2Amount be sprayed on glass fibre membrane,
After vacuum drying standby；
(4) coated film made in step (1) is pasted onto substrate, then by step (3)
In the glass fibre membrane made be pasted onto one end of coated film, absorbent paper is pasted onto coated film
The other end, glass fibre membrane away from one end of absorbent paper front paste sample pad；
(5) with cutting cutter, the big plate made in step (4) being cut into width is 3-5cm's
Wall scroll, forms foot and mouth disease and pseudorabies bigeminal Rapid detection test strip.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610465660.6A CN105954514A (en) | 2016-06-21 | 2016-06-21 | Bigeminal rapid detection test strip for foot-and-mouth disease and pseudorabies and preparation method thereof |
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| CN201610465660.6A CN105954514A (en) | 2016-06-21 | 2016-06-21 | Bigeminal rapid detection test strip for foot-and-mouth disease and pseudorabies and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645685A (en) * | 2016-11-30 | 2017-05-10 | 中国农业科学院兰州兽医研究所 | Colloidal gold test paper for testing type A foot-and-mouth disease of animals and preparation method of colloidal gold test paper |
| CN107085116A (en) * | 2017-05-16 | 2017-08-22 | 张子林 | It is a kind of to detect kit of calprotectin and preparation method thereof in human faecal mass sample |
| CN107132353A (en) * | 2017-05-16 | 2017-09-05 | 张子林 | A kind of streptococcic detection kit of B races and preparation method thereof |
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