CN105606802A - Kit for simultaneously detecting eight pig disease related antibodies and use method of kit - Google Patents
Kit for simultaneously detecting eight pig disease related antibodies and use method of kit Download PDFInfo
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- CN105606802A CN105606802A CN201510991183.2A CN201510991183A CN105606802A CN 105606802 A CN105606802 A CN 105606802A CN 201510991183 A CN201510991183 A CN 201510991183A CN 105606802 A CN105606802 A CN 105606802A
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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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention discloses a kit for simultaneously detecting eight pig disease related antibodies against CSFV (classical swine fever virus), PPV (porcine parvovirus), a PRV (porcine pseudorabies virus), PRRSV (porcine reproductive and respiratory syndrome virus), PCV2 (porcine circovirus), PEDV (porcine epidemic diarrhea virus), TGEV (transmissible gastroenteritis virus) and SIV (swine influenza virus). The kit comprises a reaction card, an ELA reagent, a color developing agent, a stop solution, a concentrated cleaning solution and a sample diluent. The invention further provides a method for simultaneously detecting eight virus related antibodies in vitro by the aid of the kit. The kit can accurately detect a CSFV antibody, a PPV antibody, a PRV antibody, a PRRSV antibody, a PCV2 antibody, a PEDV antibody, a TGEV antibody, an SIV antibody and the like and has the advantages of high sensitivity, high specificity, good accuracy, simplicity and convenience in operation, low consumption of samples, objective judgment of results and the like.
Description
Technical field
The present invention relates to detect swine disease associated antibodies kit simultaneously, relate in particular to and detect hog cholera simultaneouslyPoison (CSFV), pig parvoviral (PPV), PRV (PRV), pig blue-ear disease poison (PRRSV),Pig circular ring virus (PCV2), Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus(TGEV), 8 kinds of swine disease associated antibodies kits of swine influenza virus (SIV), described in the invention still further relates toThe using method of kit, belongs to the detection field of swine disease antibody.
Background technology
In recent years, along with the development of aquaculture, its feeding amount increased day by day, intensive,Emerge in an endless stream in large-scale cultivation field, in breeding production practice, some scale pig farm is to livestock and poultry pestilenceImmunity inoculation and daily sterilization are only focused in preventing and controlling, and to the immunoprotection effect producing after immunity inoculationFruit is relatively ignored, often think such as swine fever, pig parvoviral, porcine pseudorabies, pig blue-ear disease,The epidemic diseases such as pig circular ring virus 2 had been prevented epidemic, and just everything is just fine, and swinery just can not fall ill. Pollute journey nowadaysSpend in more serious large feeding environment, the immune effect of swinery is subject to many factors restriction, how to sentenceWhether the antibody horizontal of disconnected swinery reaches protection swinery and epidemic disease does not occur, and antibody detection is unique the most straightConnect, the most effective means, therefore strengthen antibody detection be operated in cultivation and epidemic prevention in effect be lift footWeight.
Antibody detection is the method by serological test, detects in tested serum and has by known antigensWithout corresponding specific antibody, its effect is inspection immune effect and carries out disease diagnosis. For schweineseucheThe monitoring method of sick immune level can adopt hemagglutination test (HA), hemagglutination-inhibition test (HI),Colloidal gold immunochromatographimethod technology (test strips), enzyme linked immunosorbent assay (ELISA) or Western blotting,But respectively there is its deficiency. Wherein HA, HI method for testing and detecting have in time, accurately, easy operating,The feature that basic unit is applicable, can do qualitative analysis to tested serum, can detect again number duplicate samples, both simultaneouslyThe immune state that can reflect single individuality, also can reflect the immune state of whole colony, is specially adapted toThe detection of the immune effect on scale pig farm, but the method is just for having the virus of blood clotting and resistingHealth check-up is surveyed; Colloidal gold immunochromatographimethod technology (test strips) is quick discriminating and the immune water of terrain epidemic situationFlat monitoring common technology, whole test only needs 20 minutes, easy and simple to handle, quick, accurate, sensitiveSpend high, visual result, easily judge. Colloidal gold immunochromatographimethod technology can be pointed out the existence of antiviral antibody,But single test can only detect single index, and flux is low, can not carry out specifically the kind of antiviral antibodyObjective appraisal, therefore its specific confirmation needs other technologies as Western blot, ELISA etc.Carry out secondary validation test, and the qualification of colloidal gold immunochromatographimethod technology had to certain subjectivity,Thereby may between different time points, different observers, there are differences the explanation of result; Enzyme connection is exempted fromEpidemic disease absorption method (ELISA) has that maturity is high, highly sensitive, high specificity, method of operating are easyFast, no radioactivity pollute and testing result got rid of the subjective judgement of thinking, and applied rangeEtc. lot of advantages, but exist equally single test can only detect single index, flux is low, testing costHigher, aspect the monitoring and measuring application popularization of the sick immune level of schweineseuche, existing great limitation; ImmunityTrace diagnostic reagent is the one growing up in gel electrophoresis and solid-phase immunoassay technical foundationImmune biochemical technology, has the high resolution of SDS-PAGE and the high specific of solid-phase immunoassayAdvantage with sensitiveness. But in the process of SDS-PAGE, the conformation of many antigenic determinantsMay be destroyed, can not occur with specific antibody or non-specific binding occur, be caused resultMisjudgment.
Based on above problem, urgently develop a kind of advanced, easy, can carry out similar epidemic disease together simultaneouslyThe new method of time diagnosis, improve the monitoring of the sick immune level of schweineseuche, is conducive to pig aquacultureFurther develop.
Summary of the invention
One of object of the present invention is to provide and detects CSFV (CSFV), pig parvoviral simultaneously(PPV), PRV (PRV), pig blue-ear disease poison (PRRSV), pig circular ring virus (PCV2),Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV), swine influenza virus(SIV) 8 kinds of swine disease associated antibodies kits, this kit has that susceptibility is high, high specificity, accuratelyProperty is good, easy and simple to handle, and sample consumption is few, and result judges the advantages such as objective;
Two of object of the present invention is that to detect in vitro CSFV, pig parvoviral, pig puppet mad simultaneouslyDog disease poison, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus, transmissible gastroenteritis of swineThe method of virus, 8 kinds of swine disease associated antibodies of swine influenza virus.
The present invention is achieved through the following technical solutions for achieving the above object:
First the present invention discloses and has detected CSFV (CSFV), pig parvoviral (PPV), pig simultaneouslyPseudorabies virus (PRV), pig blue-ear disease poison (PRRSV), pig circular ring virus (PCV2), pig are popular8 kinds of property diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV), swine influenza viruses (SIV)Swine disease associated antibodies kit, comprising: reaction card, elisa reagent, developer, stop buffer, concentratedCleaning solution, sample diluting liquid;
Wherein, described reaction card by slide glass and be fixed on slide glass be coated with CSFV, pig is thinSmall virus, PRV, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus,8 parallel antigen detection lines of transmissible gastro-enteritis virus, swine influenza virus and 1 are by pigThe rule nitrocellulose filter composition of the control line that forms of IgG, wherein each detection line and control line pairShould, in slide glass reaction zone in a separate reactive tank, form independently surveyed area; DescribedThe elisa reagent anti-pig IgG of rabbit that is alkali phosphatase enzyme mark; Described developer is the chloro-3-of the bromo-4-of 5-Indoles-phosphoric acid; Described stop buffer is 1 × phosphate buffer; Described sample diluting liquid is 1 ×Phosphate buffer; Described concentrated cleaning solution is 10 × phosphate buffer.
The preparation of described reaction card comprises the following steps: by CSFV, pig parvoviral, pig puppet is madDog disease poison, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus, transmissible gastroenteritis of swineVirus, swine influenza virus 8 boar disease-specific antigens and pig IgG line on nitrocellulose filter, bagQuilt, sealing, fixing slide glass, to obtain final product.
Preferably, the preparation of described reaction card comprises the following steps: (1) is coated: by the pig of 0.5ulPestivirus, pig parvoviral, PRV, pig blue-ear disease poison, pig circular ring virus, pig are popularProperty diarrhea virus, transmissible gastro-enteritis virus, swine influenza virus 8 boar disease-specific antigen and pigsIgG lines on nitrocellulose filter, 4 DEG C of coated 16-24 hour; (2) sealing: use 50-200ulConfining liquid, 37 DEG C of sealing 0.5-3 hour, with the concentrated washing lotion washing of 10 times of dilutions, after drying,Save backup in 2-8 DEG C, wherein, described confining liquid is for containing 0.05-0.5%Tween20,1-5%BSA,5-10% skimmed milk power, 5-10% sucrose, 0.01-0.1M phosphate buffer or Tris bufferingLiquid; (3) fixing slide glass: prepared detection film bar is fixed on slide glass to wherein each detectionLine is corresponding or be positioned in slide glass reaction zone in a separate reactive tank, forms independently and detectsRegion, obtains reaction card.
Described coated with antigen be CSFV, pig parvoviral, the porcine pseudorabies that deactivation is purifiedPoison, malicious, the pig circular ring virus of pig blue-ear disease, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus,The totivirus compositions such as swine influenza virus, purity reaches more than 90%.
Wherein, described CSFV, pig parvoviral, PRV, pig blue-ear disease poison, pigPCV-II, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, swine influenza virus 8 boarsThe antigen concentration of disease and pig IgG is for being 0.1-5.0ug/ml.
Preferably, described CSFV antigen concentration 2.0ug/ml, PPV Antigen Using concentration1.0ug/ml, PRV antigen concentration 4.0ug/ml, pig blue-ear disease poison antigen concentration 3.0ug/ml,Pig circular ring virus antigen concentration 1.0ug/ml, Porcine epidemic diarrhea virus antigen concentration 3.0ug/ml, pigTGE antigen concentration 5.0ug/ml, swine influenza virus antigen concentration 3.0ug/ml, pigIgG antigen concentration 1.0ug/ml.
The present invention is optimized the antigen concentration of 8 kinds of swine diseases, with the coated buffer solution of having optimized,Envelope antigen is diluted to 0.1ug/ml, 1.0ug/ml, 2.0ug/ml, 3.0ug/ml, 4.0ug/ml,Six concentration such as 5.0ug/ml, point sample is in singly examining diaphragm, respectively with CSFV, pig parvoviral,PRV, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus, pig transmissibleThe standard positive serums such as marcy agent, swine influenza virus, pig IgG carry out square formation detection, enzyme joint-trialDilution agent degree is 1:300, adopts the Q-14 of Kodak (Kodak) GTG card after hatching, develop the color, stoppingCarry out discriminatory analysis. Do not occur that with negative serum contrast spot, positive serum antibody titer are the highest, aobviousThe more than 14 minimum antigen coated concentration of intensity of colour is defined as antigen best effort concentration, finally determinesCSFV antigen concentration 2.0ug/ml, PPV Antigen Using concentration 1.0ug/ml, pseudorabiesViral antigen concentration 4.0ug/ml, pig blue-ear disease poison antigen concentration 3.0ug/ml, pig circular ring virus antigenConcentration 1.0ug/ml, Porcine epidemic diarrhea virus antigen concentration 3.0ug/ml, transmissible gastroenteritis of swine diseasePoison antigen concentration 5.0ug/ml, swine influenza virus antigen concentration 3.0ug/ml, pig IgG antigen concentration1.0ug/ml。
The present invention further discloses that described kit detects CSFV in vitro simultaneously, pig is tinyVirus, PRV, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus, pigThe method of TGE, 8 kinds of swine disease associated antibodies of swine influenza virus, comprises the following steps:(1) Sample pretreatment: venous blood sampling, centrifugation serum after solidifying; (2) application of sample: dilutionSerum, joins each reactive tank that reaction blocks, and hatches concussion; (3) wash plate: sample reaction knotShu Hou, discards reactant liquor, vibration washing; (4) add elisa reagent: in each reactive tank, addEnter elisa reagent, hatch concussion, after reaction finishes, wash film; (5) detect: each reactive tank addsNitrite ion, reacts after a period of time observed result under room temperature; (6) result is judged: a, the positive:In peep hole, there is purple line in detection line district (T) and control line district (C) simultaneously; Antibody dripsSpend highlyer, detection line (T) color is darker. B, the weak positive: in peep hole, detection line district (T)And there is purple line in control line district (C) simultaneously, but the very slight color that detection line district (T) occurs; C,Negative: in peep hole, to only have control line district (C) to occur purple line; D, inefficacy: observingIn hole, there is not purple line in control line district (C) and detection line district (T); Or detection line district only(T) there is purple line.
Preferably, the method for described kit, comprises the following steps: (1) Sample pretreatment: quietArteries and veins is got blood, puts test tube centrifugation serum after solidifying, and obtains serum sample; (2) application of sample: useSample diluting liquid, according to 1:100 dilute serum sample, adds 100ul in each reactive tank of reaction cardDilute serum sample, hatches concussion; (3) wash plate: after sample reaction finishes, discard reactant liquor,Vibration washing; (4) add elisa reagent: in each reactive tank, add 100ul elisa reagent,Hatch concussion, after reaction finishes, wash film; (5) detect: each reactive tank adds the colour developing configuringLiquid 100ul, reacts after a period of time observed result under room temperature; (6) result is judged: a, the positive:In peep hole, there is purple line in detection line district (T) and control line district (C) simultaneously; Antibody dripsSpend highlyer, detection line (T) color is darker. B, the weak positive: in peep hole, detection line district (T)And there is purple line in control line district (C) simultaneously, but the very slight color that detection line district (T) occurs; C,Negative: in peep hole, to only have control line district (C) to occur purple line; D, inefficacy: observingIn hole, there is not purple line in control line district (C) and detection line district (T); Or detection line district only(T) there is purple line.
Wherein, described in step (2), hatching concussion is at 37 DEG C of reaction 10-50min; Preferably,37 DEG C of reaction 30min; The extension rate of elisa reagent described in step (4) is 100-500 times; ExcellentThe extension rate of choosing is 300 times; Described hatch concussion be 37 DEG C reaction 10-50min; Preferably,37 DEG C of reaction 30min; The time of reacting under the room temperature described in step (5) is 5-20min; Preferably, the time is 10min.
The sensitivity and the accuracy that detect in order to improve kit, the present invention is the suitableeest anti-to serum sampleBetween seasonable, the parameter such as elisa reagent working concentration and optimal reaction time, substrate optimal reaction time entersGo optimization.
Optimization for the serum sample optimal reaction time: add after serum sample, 37 DEG C of effects10,20,30,40,50 minutes, square formation test, elisa reagent dilution factor is 1:300, through hatching,After colour developing, termination, adopt the Q-14 of Kodak (Kodak) GTG card to carry out discriminatory analysis. With the strong poison 14 that develops the colorAbove, negative serum contrast does not occur that spot is defined as the serum optimal reaction time. Final definite serumThe reaction working time is 37 DEG C of effects 30 minutes.
Optimization for elisa reagent working concentration: with sample diluting liquid dilution alkali phosphatase enzyme markThe anti-pig IgG of rabbit is to 1:100,1:200,1:300,1:400, working concentration that 1:500 is different,Respectively with CSFV, pig parvoviral, PRV, pig blue-ear disease poison, pig circular ring virus,The standard male such as Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, swine influenza virus, pig IgGProperty serum carries out square formation detection, adopts the Q-14 of Kodak (Kodak) GTG after hatching, develop the color, stoppingCard carries out discriminatory analysis. With colored intensity more than 14, negative serum contrast do not occur that spot is defined as bloodThe clear optimal reaction time. The final definite actual use of elisa reagent working concentration is 1:300.
Optimization for the elisa reagent optimal reaction time: with sample diluting liquid dilution alkaline phosphatase markThe anti-pig IgG of rabbit of note is to the working concentration of 1:300,10,20,30,40,50 points of 37 DEG C of effectsClock, respectively with CSFV, pig parvoviral, PRV, pig blue-ear disease poison, pig annulusVirus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, swine influenza virus, pig IgG etc.Standard positive serum detects square formation test, after colour developing, termination, adopts the Q-14 of Kodak (Kodak) GTGCard carries out discriminatory analysis. With colored intensity more than 14, negative serum contrast do not occur that spot is defined as bloodThe clear optimal reaction time. The final best effort time of determining elisa reagent is 30 minutes.
Optimization for the substrate optimal reaction time: add and react 5 minutes, l0 minute, 15 after substrateMinute, 20 minutes, after stopping, adopt the Q-14 of Kodak (Kodak) GTG card to carry out discriminatory analysis, determineThe best judgement time. With colored intensity more than 14, negative serum contrast do not occur that spot is defined as serumThe optimal reaction time. Final definite substrate optimal reaction time is 10 minutes.
Technical solution of the present invention compared with prior art has following beneficial effect:
Kit of the present invention can detect antibody against swine fever virus, pig parvoviral antibody, pig accuratelyPseudorabies virus antibody, pig blue-ear disease poison antibody, pig circular ring virus antibody, Porcine Epidemic DiarrheaPoison antibody, transmissible gastro-enteritis virus antibody, swine influenza virus antibody etc., have susceptibility high,High specificity, accuracy are good, easy and simple to handle, and sample consumption is few, naked eyes judgement, and without instrument, knotFruit judges the advantages such as objective has improved detection efficiency greatly, for the monitoring of the sick immune level of schweineseuche.
Brief description of the drawings
Fig. 1 kit reaction card schematic diagram;
Fig. 2 is the different antigen concentration colored intensities of the optimization of suitable working concentration: A:CSFV of antigenChange curve; The different antigen concentration colored intensity of B:PPV change curve; C:PRV differenceAntigen concentration colored intensity change curve; The different antigen concentration colored intensities of D:PRRSV change bentLine chart; The different antigen concentration colored intensity of E:PCV2 change curve; F:PEDV is synantigen notConcentration colored intensity change curve; G:TGEV; The different antigen concentrations of H:SIV (H1+H3) are aobviousIntensity of colour change curve; I: pig IgG variable concentrations colored intensity change curve;
Fig. 3 is that after the optimization of serum optimal reaction time: A:CSFV adds serum, different time is aobviousIntensity of colour change curve; B:PPV adds different time colored intensity change curve after serum;C:PRV adds different time colored intensity change curve after serum; D:PRRSV adds serumRear different time colored intensity change curve; After E:PCV2 adds serum, different time colour developing is strongDegree change curve; F:PEDV adds different time colored intensity change curve after serum; G:TGEV adds different time colored intensity change curve after serum; H:SIV (H1+H3) adds bloodDifferent time colored intensity change curve after clear; I: after pig IgG adds serum, different time develops the colorStrength Changes curve map;
Fig. 4 is the colour developing of the different elisa reagent concentration of the optimization of elisa reagent working concentration: A:CSFVStrength Changes curve map; The colored intensity change curve of the different elisa reagent concentration of B:PPV; C:The colored intensity change curve of the different elisa reagent concentration of PRV; The different elisa reagents of D:PRRSVThe colored intensity change curve of concentration; The colored intensity of the different elisa reagent concentration of E:PCV2 changesCurve map; The colored intensity change curve of the different elisa reagent concentration of F:PEDV; G:TGEVThe colored intensity change curve of different elisa reagent concentration; The different elisa reagents of H:SIV (H1+H3)The colored intensity change curve of concentration; I: pig IgG is the optimization of suitable working concentration;
Fig. 5 is the optimization of the best effort time of elisa reagent: the differential responses of A:CSFV elisa reagentThe colored intensity change curve of time; The colored intensity of B:PPV elisa reagent differential responses timeChange curve; The colored intensity change curve of C:PRV elisa reagent differential responses time; D:The colored intensity change curve of PRRSV elisa reagent differential responses time; E:PCV2 enzyme joint-trialThe colored intensity change curve of agent differential responses time; The F:PEDV elisa reagent differential responses timeColored intensity change curve; The colored intensity of G:TGEV elisa reagent differential responses time becomesChange curve map; The colored intensity change curve of H:SIV (H1+H3) elisa reagent differential responses time;I: pig IgG is the optimization of suitable working concentration;
Fig. 6 is the optimization of substrate reactions time: A:CSFV substrate differential responses time colored intensityChange curve; B:PPV substrate differential responses time colored intensity change curve; C:PRVSubstrate differential responses time colored intensity change curve; The D:PRRSV substrate differential responses time is aobviousIntensity of colour change curve; E:PCV2 substrate differential responses time colored intensity change curve; F:PEDV substrate differential responses time colored intensity change curve; The differential responses of G:TGEV substrateTime colored intensity change curve; H:SIV (H1+H3) substrate differential responses time colored intensity becomesChange curve map; I: pig IgG is the optimization of suitable working concentration.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention willMore clear along with description. It should be understood that described embodiment is only exemplary, not to the present inventionScope form any restriction. It will be understood by those skilled in the art that do not depart from of the present inventionUnder spirit and scope, can the details of technical solution of the present invention and form be modified or be replaced, but thisA little amendments or replacement all fall into protection scope of the present invention.
The preparation of embodiment 1 kit
1, the preparation of reaction card
1.1 coated
By 8 boar disease-specific antigen and the pig IgGs that contain variable concentrations of 0.5ul, with full-automaticPoint sample instrument lines on nitrocellulose filter, wherein, and CSFV (CSFV) antigen concentration 2.0ug/ml,Pig parvoviral (PPV) antigen concentration 1.0ug/ml, PRV (PRV) antigen concentration4.0ug/ml, pig blue-ear disease poison (PRRSV) antigen concentration 3.0ug/ml, pig circular ring virus (PCV2) is anti-Original content 1.0ug/ml, Porcine epidemic diarrhea virus (PEDV) antigen concentration 3.0ug/ml, pig transmissibleMarcy agent (TGEV) antigen concentration 5.0ug/ml, swine influenza virus (SIV) antigen concentration3.0ug/ml, pig IgG antigen concentration 1.0ug/ml, 4 DEG C of coated 16-24 hour. Concrete, asShown in Fig. 1, wherein, be not limited to represented form for the form of ruling, the row of various antigensRow order can be adjusted arbitrarily.
1.2 sealing
With the confining liquid of 50-200ul, (containing 0.05-0.5%Tween20,1-5%BSA, 5-10% is de-Fat milk powder, 5-10% sucrose, 0.01-0.1M phosphate buffer or Tris buffer solution), 37 DEG CSealing 0.5-3 hour, with the concentrated washing lotion washing of 10 times of dilutions. After drying, standby in 2-8 DEG C of preservationWith.
1.3 fixing slide glasses
Prepared detection film bar is fixed on slide glass, and wherein each antigen or detection line are corresponding or putBe placed in separate reactive tank of slide glass reaction zone (long 8mm, wide 4mm, high 4mm'sRectangular groove) in, form independently surveyed area, obtain reaction card.
2, the assembling of kit
1, reaction card, standard items 1 × 0.02ml, elisa reagent 1 × 3ml, sample diluting liquid 1 × 100ml,Concentrated cleaning solution 1 × 50ml, nitrite ion 1 × 30ml.
The preparation of embodiment 2 kits
1, the preparation of reaction card
1.1 coated
By 8 boar disease-specific antigen and the pig IgGs that contain variable concentrations of 0.5ul, with full-automaticPoint sample instrument lines on nitrocellulose filter, wherein, and CSFV (CSFV) antigen concentration 0.1ug/ml,Pig parvoviral (PPV) antigen concentration 0.1ug/ml, PRV (PRV) antigen concentration0.1ug/ml, pig blue-ear disease poison (PRRSV) antigen concentration 0.1ug/ml, pig circular ring virus (PCV2) is anti-Original content 0.1ug/ml, Porcine epidemic diarrhea virus (PEDV) antigen concentration 0.1ug/ml, pig transmissibleMarcy agent (TGEV) antigen concentration 0.1ug/ml, swine influenza virus (SIV) antigen concentration0.1ug/ml, pig IgG antigen concentration 0.1ug/ml, 4 DEG C of coated 16-24 hour. Concrete, asShown in Fig. 1, wherein, be not limited to represented form for the form of ruling, the row of various antigensRow order can be adjusted arbitrarily.
1.2 sealing
With the confining liquid of 50-200ul, (containing 0.05-0.5%Tween20,1-5%BSA, 5-10% is de-Fat milk powder, 5-10% sucrose, 0.01-0.1M phosphate buffer or Tris buffer solution), 37 DEG CSealing 0.5-3 hour, with the concentrated washing lotion washing of 10 times of dilutions. After drying, standby in 2-8 DEG C of preservationWith.
1.3 fixing slide glasses
Prepared detection film bar is fixed on slide glass, and wherein each antigen or detection line are corresponding or putBe placed in separate reactive tank of slide glass reaction zone (long 8mm, wide 4mm, high 4mm'sRectangular groove) in, form independently surveyed area, obtain reaction card.
2, the assembling of kit
1, reaction card, standard items 1 × 0.02ml, elisa reagent 1 × 3ml, sample diluting liquid 1 × 100ml,Concentrated cleaning solution 1 × 50ml, nitrite ion 1 × 30ml.
The preparation of embodiment 3 kits
1, the preparation of reaction card
1.1 coated
By 8 boar disease-specific antigen and the pig IgGs that contain variable concentrations of 0.5ul, with full-automaticPoint sample instrument lines on nitrocellulose filter, wherein, and CSFV (CSFV) antigen concentration 5.0ug/ml,Pig parvoviral (PPV) antigen concentration 5.0ug/ml, PRV (PRV) antigen concentration5.0ug/ml, pig blue-ear disease poison (PRRSV) antigen concentration 5.0ug/ml, pig circular ring virus (PCV2) is anti-Original content 5.0ug/ml, Porcine epidemic diarrhea virus (PEDV) antigen concentration 5.0ug/ml, pig transmissibleMarcy agent (TGEV) antigen concentration 5.0ug/ml, swine influenza virus (SIV) antigen concentration5.0ug/ml, pig IgG antigen concentration 5.0ug/ml, 4 DEG C of coated 16-24 hour. Concrete, asShown in Fig. 1, wherein, be not limited to represented form for the form of ruling, the row of various antigensRow order can be adjusted arbitrarily.
1.2 sealing
With the confining liquid of 50-200ul, (containing 0.05-0.5%Tween20,1-5%BSA, 5-10% is de-Fat milk powder, 5-10% sucrose, 0.01-0.1M phosphate buffer or Tris buffer solution), 37 DEG CSealing 0.5-3 hour, with the concentrated washing lotion washing of 10 times of dilutions. After drying, standby in 2-8 DEG C of preservationWith.
1.3 fixing slide glasses
Prepared detection film bar is fixed on slide glass, and wherein each antigen or detection line are corresponding or putBe placed in separate reactive tank of slide glass reaction zone (long 8mm, wide 4mm, high 4mm'sRectangular groove) in, form independently surveyed area, obtain reaction card.
2, the assembling of kit
1, reaction card, standard items 1 × 0.02ml, elisa reagent 1 × 3ml, sample diluting liquid 1 × 100ml,Concentrated cleaning solution 1 × 50ml, nitrite ion 1 × 30ml.
The using method of embodiment 4 kits
Sample requirement: venous blood sampling 2-5ml, puts test tube centrifugation serum after solidifying, serum sampleThis preservation 2-8 DEG C, the sample that can not test in 24 hours should be put-20 DEG C, and significant hemolysis, has waddingShape thing or the serum specimen going mouldy can affect test.
1, application of sample
With sample diluting liquid (phosphate buffer), according to 1:100 dilute serum sample, reaction blocksEach reactive tank in add 100ul dilute serum sample, put into and hatch oscillator. 37 DEG C of reactions 30Minute.
2, wash plate
After sample reaction finishes, discard reactant liquor, vibration washing 3 × 5min.
3, add elisa reagent
300 times of dilutions of antibody of the anti-pig IgG of rabbit of alkali phosphatase enzyme mark, in each reactive tankAdd 100ul, put into incubation oscillator, 37 DEG C are reacted 30 minutes. After finishing, reaction washes film.
4, detect
Each reactive tank adds the nitrite ion 100ul configuring, room temperature 10min observed result.
5, result is judged
A, the positive: in peep hole, detection line district (T) and control line district (C) occurs purple simultaneouslyColo(u)r streak. Antibody titer is higher, and detection line (T) color is darker.
B, the weak positive: in peep hole, detection line district (T) and control line district (C) goes out simultaneouslyExisting purple line, but the very slight color that detection line district (T) occurs;
C, feminine gender: in peep hole, only have control line district (C) to occur purple line;
D, inefficacy: in peep hole, control line district (C) and detection line district (T) do not occurPurple line; Or there is purple line in detection line district (T) only.
The using method of embodiment 5 kits:
Sample requirement: venous blood sampling 2-5ml, puts test tube centrifugation serum after solidifying, serum sampleThis preservation 2-8 DEG C, the sample that can not test in 24 hours should be put-20 DEG C, and significant hemolysis, has waddingShape thing or the serum specimen going mouldy can affect test.
1, application of sample
With sample diluting liquid (phosphate buffer), according to 1:100 dilute serum sample, reaction blocksEach reactive tank in add 100ul dilute serum sample, put into and hatch oscillator. 37 DEG C of reactions 10Minute.
2, wash plate
After sample reaction finishes, discard reactant liquor, vibration washing 3 × 5min.
3, add elisa reagent
100 times of dilutions of antibody of the anti-pig IgG of rabbit of alkali phosphatase enzyme mark, in each reactive tankAdd 100ul, put into incubation oscillator, 37 DEG C are reacted 10 minutes. After finishing, reaction washes film.
4, detect
Each reactive tank adds the nitrite ion 100ul configuring, room temperature 5min observed result.
5, result is judged
A, the positive: in peep hole, detection line district (T) and control line district (C) occurs simultaneouslyPurple line. Antibody titer is higher, and detection line (T) color is darker.
B, the weak positive: in peep hole, detection line district (T) and control line district (C) goes out simultaneouslyExisting purple line, but the very slight color that detection line district (T) occurs;
C, feminine gender: in peep hole, only have control line district (C) to occur purple line;
D, inefficacy: in peep hole, control line district (C) and detection line district (T) do not occurPurple line; Or there is purple line in detection line district (T) only.
The using method of embodiment 6 kits:
Sample requirement: venous blood sampling 2-5ml, puts test tube centrifugation serum after solidifying, serum sampleThis preservation 2-8 DEG C, the sample that can not test in 24 hours should be put-20 DEG C, and significant hemolysis, has waddingShape thing or the serum specimen going mouldy can affect test.
1, application of sample
With sample diluting liquid (phosphate buffer), according to 1:100 dilute serum sample, reaction blocksEach reactive tank in add 100ul dilute serum sample, put into and hatch oscillator. 37 DEG C of reactions 50Minute.
2, wash plate
After sample reaction finishes, discard reactant liquor, vibration washing 3 × 5min.
3, add elisa reagent
500 times of dilutions of antibody of the anti-pig IgG of rabbit of alkali phosphatase enzyme mark, in each reactive tankAdd 100ul, put into incubation oscillator, 37 DEG C are reacted 50 minutes. After finishing, reaction washes film.
4, detect
Each reactive tank adds the nitrite ion 100ul configuring, room temperature 20min observed result.
5, result is judged
A, the positive: in peep hole, detection line district (T) and control line district (C) occurs simultaneouslyPurple line. Antibody titer is higher, and detection line (T) color is darker.
B, the weak positive: in peep hole, detection line district (T) and control line district (C) goes out simultaneouslyExisting purple line, but the very slight color that detection line district (T) occurs;
C, feminine gender: in peep hole, only have control line district (C) to occur purple line;
D, inefficacy: in peep hole, control line district (C) and detection line district (T) do not occurPurple line; Or there is purple line in detection line district (T) only.
The optimization of experimental example 1 relevant parameters
1, experimental technique
1.1 antigens are the optimization of suitable working concentration
With the coated buffer solution of having optimized, envelope antigen is diluted to 0.1ug/ml, 1.0ug/ml,2.0ug/ml, 3.0ug/ml, 4.0ug/ml, six concentration such as 5.0ug/ml, point sample is in singly examining diaphragm,Respectively with CSFV, pig parvoviral, PRV, pig blue-ear disease poison, pig circular ring virus,The standard male such as Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, swine influenza virus, pig IgGProperty serum carries out square formation detection, and elisa reagent dilution factor is 1:300, through hatching, develop the color, stoppingThe Q-14 of rear employing Kodak (Kodak) GTG card carries out discriminatory analysis. This GTG card is from vain to black totally 20Individual contrast, wherein 0 is white, and 20 is black, and the larger color of numeral is the darkest, and this test will detectLine colour developing and diagnosis diaphragm background color are compared with GTG card respectively, the strong poison of colour developing develop the color for detection line andThe aberration of diagnosis diaphragm background color, aberration is larger, and the strong poison that develops the color is stronger. The intensity of colour developing is oneDetermine in scope to be directly proportional to coated antigen amount, but along with the increase of envelope antigen concentration, colour developing by forceDegree reduces on the contrary, does not occur that spot, positive serum antibody titer are the highest, colour developing with negative serum contrastThe more than 14 minimum antigen coated concentration of intensity is defined as antigen best effort concentration. Wherein, optimizeGood coated buffer solution is PBST (pH value 7.4), in table 1.
Table 1PBST formula (1L)
The optimization of 1.2 serum optimal reaction times
Add after serum, 37 DEG C of effects 10,20,30,40,50 minutes, square formation test, enzymeJoint-trial dilution agent degree is 1:300, adopts the Q-14 of Kodak (Kodak) ash after hatching, develop the color, stoppingRank card carries out discriminatory analysis. The intensity of colour developing is directly proportional to the reaction time of serum within the specific limits,But along with the increase of time, the color of detection line is constant, and background color deepens, with colored intensity 14Above, negative serum contrast does not occur that spot is defined as the serum optimal reaction time.
The optimization of 1.3 elisa reagent working concentrations
With the anti-pig IgG of rabbit of sample diluting liquid dilution alkali phosphatase enzyme mark to 1:100,1:200,1:300,1:400, the working concentration that 1:500 is different, respectively with CSFV, pig parvoviral,PRV, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus, pig transmissibleThe standard positive serums such as marcy agent, swine influenza virus, pig IgG carry out square formation detection, through hatching,After colour developing, termination, adopt the Q-14 of Kodak (Kodak) GTG card to carry out discriminatory analysis. Colored intensity is oneDetermine to be directly proportional to elisa reagent working concentration in scope, but the increase of elisa reagent working concentration, detection lineColor constant, background color deepens, with colored intensity more than 14, negative serum contrast do not occurSpot is defined as the serum optimal reaction time.
The optimization of the best effort time of 1.4 elisa reagents
Dense to the work of 1:300 with the anti-pig IgG of rabbit of sample diluting liquid dilution alkali phosphatase enzyme markDegree, 37 DEG C of effects 10,20,30,40,50 minutes, respectively with CSFV, the tiny disease of pigPoison, PRV, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus, pig passThe standard positive serums such as metachromia marcy agent, swine influenza virus, pig IgG detect square formation test, warpAfter colour developing, termination, adopt the Q-14 of Kodak (Kodak) GTG card to carry out discriminatory analysis. Colored intensity is oneDetermine in scope to be directly proportional to the reaction time of elisa reagent, but along with the increase of time, the face of detection lineLook constant, and background color deepens, with colored intensity more than 14, spot do not appear in negative serum contrastBe defined as the serum optimal reaction time.
The optimization of 1.5 substrate reactions times
Add react after substrate 5 minutes, l0 minute, 15 minutes, 20 minutes, after stopping, adopt KeReach (Kodak) Q-14 GTG card and carry out discriminatory analysis, determine the best judgement time. The intensity of colour developing existsIs directly proportional to the substrate reactions time in certain limit, but along with the increase of time, the color of detection line is notBecome, background color deepens, with colored intensity more than 14, negative serum contrast do not occur that spot is definiteFor the serum optimal reaction time.
2. experimental result
2.1 antigens are the optimization of suitable working concentration
Different antigen concentration colored intensities change in table 2 and Fig. 2, from table 2 and Fig. 2, can find out,8 kinds of antigen at colored intensity more than 14 and the minimum antigen coated concentration of pig IgG are respectively swine feverVirus (CSFV) antigen concentration 2.0ug/ml, pig parvoviral (PPV) antigen concentration 1.0ug/ml, pigPseudorabies virus (PRV) antigen concentration 4.0ug/ml, pig blue-ear disease poison (PRRSV) antigen concentration3.0ug/ml, pig circular ring virus (PCV2) antigen concentration 1.0ug/ml, Porcine epidemic diarrhea virus (PEDV)Antigen concentration 3.0ug/ml, transmissible gastro-enteritis virus (TGEV) antigen concentration 5.0ug/ml, pig streamInfluenza Virus (SIV) antigen concentration 3.0ug/ml, pig IgG antigen concentration 1.0ug/ml. Therefore determine above-mentionedThe concentration of each antigen is best coated concentration.
Table 2 is diagnosed the suitableeest working concentration of diaphragm antigen (elisa reagent working concentration is 1:300)
The optimization of 2.2 serum optimal reaction times
The colored intensity of every kind of detection line and serum optimal reaction time the results are shown in Table 3 and Fig. 3, comprehensiveEach virus, at different time colored intensity, finally determines that the seroreaction working time is 30 minutes.
The optimization of table 3 serum optimal reaction time
The optimization of 2.3 elisa reagent working concentrations
What elisa reagent working concentration detected effect the results are shown in Table 4 and Fig. 4, and comprehensive each virus is at different enzymesThe colored intensity situation of change of joint-trial agent concentration, finally determines that elisa reagent working concentration is actual to make recruitmentBe 1:300 as concentration.
The suitableeest working concentration of table 4 elisa reagent
The optimization of the best effort time of 2.4 elisa reagents
The elisa reagent optimal reaction time the results are shown in Table 5 and Fig. 5, and comprehensive each virus is in elisa reagent differenceThe situation of change of the colored intensity in reaction time, the best effort time of determining elisa reagent is 30 pointsClock.
The optimization of the best effort time of table 5 elisa reagent
The optimization of 2.5 substrate reactions times
The substrate optimal reaction time the results are shown in Table 6 and Fig. 6, and comprehensive each virus is in the time of substrate differential responsesBetween colored intensity situation of change, finally determine the substrate optimal reaction time be 10 minutes.
The optimization of table 6 substrate reactions time
Experimental example 2 kit of the present invention detects test
1, test method
1.1 specific test
To diagnose diaphragm and CSFV (CSFV), pig parvoviral (PPV), PRV(PRV), pig blue-ear disease poison (PRRSV), pig circular ring virus (PCV2), Porcine epidemic diarrhea virus(PEDV), the positive serum such as transmissible gastro-enteritis virus (TGEV), swine influenza virus (SIV) isUnder suitable condition, act on, detect whether there is cross reaction, determine its specificity.
1.2 Clinical detection tests
Inspection center of Harbin Veterinary Medicine Inst., China Academy of Agriculture provides 100 parts of blood serum samples, inspectionMeasured center application U.S. Ai Deshi (IDEXX) kit detects serum antibody, and wherein 88 parts of serum containAntibody against swine fever virus, 96 parts of serum contain porcine pseudorabies virus containing pig parvoviral antibody, 78 parts of serumAntibody, 72 parts of serum contain pig circular ring virus antibody, 36 parts of blood containing pig blue-ear disease poison antibody, 86 parts of serumClear Porcine epidemic diarrhea virus antibody, the 40 parts of serum of containing are containing transmissible gastro-enteritis virus antibody, 42Part serum is containing swine influenza virus antibody. Adopt kit of the present invention (embodiment 1 is prepared) to detect,The strict kit description of pressing operates.
2 result of the tests
2.1 specific test
Result shows: pewter spot (table 7) only appears in each antigen between corresponding antigens, antibody,Show stronger specificity.
Table 7 specific test
Note: "-" represents that result is negative, "+" represents that result is negative.
2.2 Clinical detection tests
Testing result and U.S. Ai Deshi (IDEXX) kit testing result have good coincidence rate,Result of the test is in table 8.
The colour developing result that table 8 detects 100 parts of serum
To sum up result of the test is visible, kit of the present invention can detect accurately antibody against swine fever virus,Pig parvoviral antibody, porcine pseudorabies virus antibody, pig blue-ear disease poison antibody, pig circular ring virus resistBody, Porcine epidemic diarrhea virus antibody, transmissible gastro-enteritis virus antibody, swine influenza virus antibodyDeng, having that susceptibility is high, high specificity, accuracy be good, easy and simple to handle, sample consumption is few, resultJudge the advantages such as objective.
Claims (10)
1. detect CSFV, pig parvoviral, PRV, pig blue-ear disease poison, pig simultaneouslyPCV-II, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, 8 kinds of swine diseases of swine influenza virusAssociated antibodies kit, comprising: reaction card, elisa reagent, developer, stop buffer, thickening and washingLiquid, sample diluting liquid, is characterized in that, described reaction card is by slide glass and be fixed on being coated with on slide glassThere are CSFV, pig parvoviral, PRV, pig blue-ear disease poison, pig circular ring virus, pig8 parallel antigens of epidemic diarrhea virus, transmissible gastro-enteritis virus, swine influenza virus detectLine and 1 are made up of the rule nitrocellulose filter of the control line that forms of pig IgG, wherein each detection line, form independently and detect corresponding in a separate reactive tank in slide glass reaction zone with control lineRegion.
2. according to kit claimed in claim 1, it is characterized in that: described elisa reagent isThe anti-pig IgG of rabbit of alkali phosphatase enzyme mark; Described developer is 5-bromo-4-chloro-3-indolylphosphate;Described stop buffer is 1 × phosphate buffer; Described concentrated cleaning solution is 10 × phosphate buffer;Described sample diluting liquid is 1 × phosphate buffer.
3. according to the kit described in claim 1 or 2, it is characterized in that the preparation of described reaction cardComprise the following steps: by CSFV, pig parvoviral, PRV, pig blue-ear disease poison,Pig circular ring virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, swine influenza virus 8 boarsDisease-specific antigen and pig IgG line respectively on nitrocellulose filter, coated, sealing, fixing carryingSheet, to obtain final product.
4. according to kit claimed in claim 3, it is characterized in that: described CSFV, pig are thinSmall virus, PRV, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus,The antigen concentration of transmissible gastro-enteritis virus, 8 kinds of swine diseases of swine influenza virus and pig IgG is 0.1-5.0ug/ml。
5. according to kit claimed in claim 4, it is characterized in that: described CSFV antigen is denseDegree 2.0ug/ml, PPV Antigen Using concentration 1.0ug/ml, PRV antigen concentration4.0ug/ml, pig blue-ear disease poison antigen concentration 3.0ug/ml, pig circular ring virus antigen concentration 1.0ug/ml,Porcine epidemic diarrhea virus antigen concentration 3.0ug/ml, transmissible gastro-enteritis virus antigen concentration5.0ug/ml, swine influenza virus antigen concentration 3.0ug/ml, pig IgG antigen concentration 1.0ug/ml.
6. described in an application rights requirement 1-2, kit detects CSFV, pig in vitro simultaneouslyParvovirus, PRV, pig blue-ear disease poison, pig circular ring virus, Porcine epidemic diarrhea virus,The method of transmissible gastro-enteritis virus, 8 kinds of swine disease associated antibodies of swine influenza virus, is characterized in that,Comprise the following steps: (1) Sample pretreatment: separation of serum; (2) application of sample: dilute serum, addsEnter to each reactive tank of reaction card, hatch concussion; (3) wash plate: after sample reaction finishes, discardReactant liquor, vibration washing; (4) add elisa reagent: in each reactive tank, add elisa reagent,Hatch concussion, after reaction finishes, wash film; (5) detect: each reactive tank adds nitrite ion, under room temperatureAfter reaction a period of time, observed result; (6) result is judged: a, the positive: in peep hole, and inspectionThere is purple line in survey line district (T) and control line district (C) simultaneously; Antibody titer is higher, detection line (T)Color is darker; B, the weak positive: in peep hole, detection line district (T) and control line district (C) is sameTime there is purple line, but detection line district (T) occur very slight color; C, feminine gender: in peep hole,Only have control line district (C) to occur purple line; D, inefficacy: in peep hole, control line district (C)There is not purple line with detection line district (T); Or there is purple line in detection line district (T) only.
7. it is characterized in that in accordance with the method for claim 6: step is incubated described in (2)Educating concussion is at 37 DEG C of reaction 10-50min; Preferably, 37 DEG C of reaction 30min.
8. it is characterized in that in accordance with the method for claim 6: enzyme described in step (4)The extension rate of joint-trial agent is 100-500 times; Preferred extension rate is 300 times.
9. it is characterized in that in accordance with the method for claim 6: step is incubated described in (4)Educating concussion is at 37 DEG C of reaction 10-50min; Preferably, 37 DEG C of reaction 30min.
10. it is characterized in that in accordance with the method for claim 6: described in step (5)The time of reacting under room temperature is 5-20min; Preferably, the time is 10min.
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