CN202814986U - Test paper strip for distinguishing foot and mouth disease virus infection and vaccine immune animals by one step - Google Patents

Abstract

The utility model relates to a test paper strip for rapidly distinguishing foot and mouth disease virus infection and vaccine immune animals, which comprises a support layer and an adsorption layer, wherein the adsorption layer is attached on the support layer; the adsorption layer sequentially comprises an adsorption fiber layer, a gold-labeled antibody fiber layer, a cellulose membrane layer and a water absorbing material layer arranged at a handle end from a test end; the cellulose membrane layer is provided with detection blot and comparison blot; staphylococcal protein A labeled by colloidal gold is adsorbed on the gold-labeled antibody fiber layer; the detection blot is expressed by Escherichia coli expression system and printed by one or two in purified by pig foot-and-mouth disease virus structural protein VP1 antigen and non-structural protein 3ABC antigen; and the comparison blot is printed by goat or rabbit anti-SPA IgG antibody solution. The test paper strip is high in specificity and sensitivity, simple, convenient, visual, accurate, wide in application scope and low in cost, and can be used for detecting at any time and any place by professionals and non-professionals, thus being easy to popularize and apply.

Description

One step was differentiated the test strips of mouth disease virus infection and vaccine immunity animal

Technical field

The utility model relates to a kind of utensil that detects foot and mouth disease virus, particularly relates to the test strips of a kind of quick discriminating mouth disease virus infection and vaccine immunity animal.

Background technology

Aftosa (Foot-and-mouth-disease, FMD) is to cause a kind of strong infectiousness epidemic disease artiodactylous by foot and mouth disease virus (FMDV).FMDV has the characteristics such as polytypism, changeableness, host's popularity, contagiousness be extremely strong, in case morbidity namely is popular, great outburst.The outburst of FMD and popularly often bring about great losses to Infected regions, except the direct economic loss that animal dead causes, also have that the output of during one's sickness meat and the damp production of milk of animal, recover rear meat and milk is long-term to descend and plant with being worth the greater loss such as forfeiture.More seriously, FMD is a kind of deadly infectious disease, has extremely strong infectiousness, for the same group motion thing of morbidity animal and contact cause of disease, must isolate strictly, block, forbid that animal is moved with the livestock products listing etc.Therefore, FMD causes the livestock products foreign trade of Infected regions even morbidity country to stop, and except causing huge economic loss, also produces certain political fallout.

Vaccine inoculation is the main policies of the control FMD of developing country, because FMD is intrinsic, present vaccine both domestic and external can only protect immune animal that FMD does not occur, but this method can not prevent that immune animal from the infection of FMDV occuring again.In the immune animal group in epidemic-stricken area, the animal that some is arranged is the virus carrier, and these animals might continue outside toxin expelling, but forms the virus long-term existence of persistent infection in ox, sheep body.The animal of these subclinical infections or band poison not only may cause new epidemic situation, also provides environment for virus variation and the new strain of generation.Therefore, FMDV vaccine immunity animal's antibody level and how to distinguish immune animal and the virus infections animal be control FMDV key issue.

The FMDV vaccine that uses at present mainly is inactivated vaccine and synthetic peptide vaccine.Behind the inactivated vaccine immune animal, do not have virus multiplication in the animal body, in the viral purification process, removed most non-structural protein (NSP) in the production of vaccine, there has not been the expression of non-structural protein, thereby in the immune animal body, just do not have the generation of non-structural protein antibody, polypeptide vaccine itself is exactly a Main Antigenic on the FMDV structural proteins VP1, does not more have the existence of non-structural protein.And in the animal body that FMDV infects, the expression of a large amount of non-structural proteins is arranged in viral assembling process, stimulate body to produce corresponding non-mechanism protein antibodies.The structural proteins antibody test can monitor animal immune state and antibody produce level, non-structural protein antibody test technology provides foundation for distinguishing infection animal and immune animal, the purpose that the combination of structural proteins and non-structural protein detection technique can reach a step detection FMDV immune antiboidy level and distinguish FMDV infection animal and vaccine immunity animal.

The method of at present laboratory detection FMDV antibody has: (1) virus is separated: isolate virus from the pathological material of disease of infected animal, again the virus that is separated to is carried out culture identification and make diagnosis, determine whether that FMDV infects.This technology accurately and reliably, but susceptibility is poor, and has complement activity in some sample, impact detects effect, and needs special laboratory.(2) virus neutralization tests: utilize the interaction of virus and specific serum neutralizing antibody, thereby make virus lose infection ability to susceptible animal and sensitive cells, this method must be used live virus, and common lab can not operate.Enzyme linked immunosorbent assay (ELISA): mainly contain the non-structural protein ELISA(I-ELISA that distinguish to infect with immunity) and the LPB-ELISA of the antibody level of serum of detection vaccine immunity animal (3).Have the advantages such as special, responsive when the ELISA method is measured, but complicated operation, time-consuming, effort need specific instrument and equipment and professional and technical personnel, are difficult to the penetration and promotion in basic unit.Simultaneously, the LPB-ELISA method needs the participation of inactivation of viruses, therefore, also has the incomplete or viral escape equivalent risk of inactivation of virus.In addition, along with the applying of pig O type FMD synthetic peptide vaccine, progressively occupied the principal market in recent years, traditional LPB-ELISA is difficult to polypeptide vaccine antibody is made accurately testing result.Therefore, study a kind of easy fast, a step can detect the real-time online detection technique of vaccine immunity and virus infections, and is extremely important and urgent.

The utility model content

The technical problems to be solved in the utility model: overcome in the prior art and to detect the defective that antibodies against foot-and-mouth disease virus and vaccine antibody exist, provide a kind of high specificity, susceptibility high, easy, differentiate the immune-gold labeled test strips of antibodies against foot-and-mouth disease virus and vaccine antibody fast.

The technical solution of the utility model:

One step was differentiated the test strips of mouth disease virus infection and vaccine immunity animal, contain supporting layer and adsorbed layer, adsorbed layer is attached on the supporting layer, adsorbed layer is followed successively by sample adsorbing fiber layer from test lead, gold labeling antibody fibrage, the absorbent material layer of cellulose rete and handle end, be provided with detection trace and contrast trace at the cellulose rete, the staphylococcal protein A of colloid gold label is arranged is SPA in absorption on the described golden labeling antibody fibrage, described detection trace is expressed with escherichia expression system and the swine foot-and-mouth disease virus structural proteins VP1 antigen of purifying and one or both printings in the nonstructural protein 3A BC antigen, and the contrast trace is printed with the IgG antibody of sheep or the anti-SPA of rabbit.

Described supporting layer is made with the hard plastic bar or the cardboard bar that do not absorb water; Sample adsorbing fiber layer is made with glass wool, nylon fiber or dacron; Gold labeling antibody fibrage is made with glass wool.

Described cellulose rete is made with nitrocellulose filter, cellulose membrane, carboxymethyl cellulose film or polyvinylidene fluoride PVDF cellulose membrane; Described absorbent material layer is made with thieving paper.

Contain one/on the described cellulose rete when detecting trace, the spread pattern that detects trace, contrast trace be " ||", in " 10 ", " ┬ ┬ ", " ┴ ┴ ", " ├ ├ ", " ┤ ┤ " and " ● ● " any; Contain two/on the cellulose rete when detecting trace, the spread pattern that detects trace, contrast trace be " || |", " 10 ", " ┬ ┬ ┬ ", " ┴ ┴ ┴ ", " ├ ├ ├ ", " ┤ ┤ ┤ " and " ● ● ● " in any.

Be coated with diaphragm at described sample adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer, the diaphragm corresponding with golden labeling antibody fibrage intersection at sample adsorbing fiber layer is printed with the sample mark line.

Positive beneficial effect of the present utility model:

1, detection specificity is strong, susceptibility is high: the utility model test strips is prepared from as the basis take the SPA of colloid gold label, form without covalent bond between gold grain among the gold mark SPA and antigen or the SPA molecule, the two combines by the Van der Waals force between the charges of different polarity, collaurum affects very little on the swine foot-and-mouth disease virus structural proteins VP1 antigen of escherichia expression system expression and purifying and the specificity of nonstructural protein 3A BC antigen, do not affect the combination of SPA and antibody and the combination of antibody and antigen yet, and have higher mark rate.Therefore, test strips of the present utility model has higher specificity and susceptibility, can detect the corresponding antibodies albumen of 2 nanograms.

2, easy and simple to handle, quick: as to need not additional Other Instruments and reagent in the ELISA test strip process of the present utility model, as long as its test lead was inserted in the serum to be checked about 30 seconds, wait about 5 minutes and can judge testing result.

3, intuitive display, accurately as a result: test strips is to show that henna detection trace and contrast trace are as the positive and the negative marker that detect, namely on the cellulose rete, only show a brownish red contrast trace C, be illustrated in the tested serum and both infected antibody also without FMDV vaccine immunity antibody without FMDV, namely tested animal infects also defective without FMDV immunity or immunity without FMDV; If show that at the cellulose rete brownish red contrast trace C and two brownish reds detect trace P, J, represent that then tested animal has FMDV to infect; If show that at the cellulose rete brownish red contrast trace C and a brownish red detect trace P, then are illustrated in and contain FMDV vaccine immunity antibody in the tested serum; It is directly perceived, accurate, simple and clear that the result judges, is not prone to false negative and false-positive erroneous judgement.

4, production system is reliable: the detection trace in the utility model adopts escherichia expression system as production system, the research of this expression system is comparatively deep, through repeatedly checking, condition of culture is simple during as production system, genetic manipulation is easy, shorter from being building up to the product purification cycle, cost is low, and output is high, is suitable for industrial applications.

5, small investment, cost is low: use the utility model test strips not need to join in addition Other Instruments, equipment and reagent, save large measuring appratus, equipment and additive reagent expense; Article one, test strips once can detect one or both antibody, and specialty and layman all can carry out real-time online whenever and wherever possible and detect, and need not to pay expert diagnosis expense and correlative charges, can save testing cost, reduces testing cost.

6, applied range, be convenient to promote: test strips of the present utility model one-tenth simple to operate " single step ", " foolproof ", and be convenient for carrying and preserve, can satisfy different levels personnel's needs, comprise professional chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, livestock products processing, intensive culture to individual cultivation etc., have wide market outlook and economical, societal benefits preferably.

Description of drawings

The structural representation of Fig. 1 the utility model test strips;

The plan structure synoptic diagram of Fig. 2 the utility model test strips.

Embodiment

Following examples are in order to further specify the utility model, not represent restriction of the present utility model.Wen Zhongru does not specify that percentage composition wherein is the quality percentage composition.

The test strips for preparing a step discriminating mouth disease virus infection and vaccine immunity animal, prepare first escherichia expression system and express also swine foot-and-mouth disease virus structural proteins VP1 antigen and the nonstructural protein 3A BC antigen of purifying, and the IgG antibody of anti-SPA, then prepare golden labeling antibody fibrage, detect trace and contrast trace.

1, the preparation of the IgG antibody of goat-anti or the anti-SPA of rabbit:

Consumption with per kilogram of body weight 50 microgram SPA, through the negative Healthy Sheep of subcutaneous and intramuscular injection (or rabbit) 3～4 times, last immunity posterior vein blood sampling in 20 days is measured its serum antibody titer more than 1:2000 with ELISA, its hyper-immune serum is collected in heart blood sampling or arteria carotis bloodletting.Get 1 part of serum and add 2 parts of PBS liquid mixings, add isopyknic saturated ammonium sulfate liquid mixing, put in 4 ℃ of refrigerators 2 hours, 4 ℃, 10000 rev/mins centrifugal 15 minutes, abandon supernatant; With an amount of PBS liquid dissolution precipitation, adding saturated ammonium sulfate solution to its ultimate density is 33%, put in 4 ℃ of refrigerators 2 hours, and under 4 ℃, 10000 rev/mins conditions centrifugal 15 minutes, abandoned supernatant, with a small amount of PBS liquid dissolution precipitation, put in 4 ℃ of refrigerators with PBS liquid dialyzed overnight, change liquid 2～3 times, centrifugal 15 minutes 4 ℃, 10000 rev/mins, collect supernatant, measure its protein concentration with ultraviolet spectrophotometer.The IgG antibody of the anti-SPA of sheep (or rabbit) that extracts with the saturated ammonium sulfate method is for the preparation of the contrast trace.

2, the preparation of the swine foot-and-mouth disease virus structural proteins VP1 antigen of Bacillus coli expression and purifying and nonstructural protein 3A BC antigen:

(1) swine foot-and-mouth disease virus structural proteins VP1 Expression in Escherichia coli and purifying

According to a pair of Auele Specific Primer of O type FMDV VP1 templet gene sequences Design: in upstream primer, introduce restriction enzyme site BamHI introduces restriction enzyme site in the downstream primer HindIII.Take the recombinant plasmid pMD18T-VP1 that contains the VP1 gene as template, obtain the VP1 gene through the PCR amplification.Wherein the PCR reaction system is: template pMD18T-VP1 0.5 microlitre, upstream primer 0.8 microlitre, downstream primer 0.8 microlitre, Ex Taqase premix enzyme 25 microlitres, distilled water 22.9 microlitres.Amplification program: 94 ℃ of denaturations 4 minutes, circulation primary; 94 ℃ of sex change 45 seconds, 60 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, and circulated altogether 35 times; 72 ℃ were extended 10 minutes.The VP1 gene warp of PCR amplification gained BamHI and HindThe digestion of III double digestion is cloned in the PET-28a carrier, transform coli strain, recombinant bacterium is cultured to OD600 reaches at 0.6 o'clock in containing the LB nutrient culture media of ampicillin (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, ampicillin mg/L), add isopropyl-β-D-sulfo-galactopyranoside derivant, making its final concentration is 0.5mmol/L, 37 ℃ of abduction deliverings 6 hours, it is centrifugal to express bacterium liquid, the results bacterial precipitation.

Thalline is resuspended in 20 milliliters of damping fluids after (damping fluid composition: 10 mmol/L Tris-HCl pH, 8.0,150 mmol/L NaCl, 10 mmol/L EDTA, 10 % glycerine, 2 mmol/L DTT, 0.5 mmol/L PMSF) ultrasonication, centrifugal collecting precipitation washs inclusion body 5 times with washing lotion (washing lotion composition: the Triton-100 of 1 % volume, 50 mmol/L Tris-HCl pH, 8.0,100 mmol/L NaCl, 10 mmol/L EDTA, 2 mmol/L DTT); Then will be deposited in 1 time (suspending liquid composition: 50 mmol/L Tris-HCl pH, 8.0,100 mmol/L NaCl, 10 mmol/L EDTA, 2 mmol/L DTT) of washing in 20 milliliters of suspending liquid, last centrifugation is the inclusion body behind the purifying.

It is (denaturant composition: urea 8mol/L, NaCl 0.5mol/L, Tris-HCl 20mmol/L, EDTA 20mmol/L, 2% SDS) in 8.0 the denaturant that inclusion body behind the purifying is dissolved in pH, in 4 ℃ of stir abouts 10 hours, the dissolving sex change, centrifugal removal insolubles obtains metaprotein; 90 milliliters of metaproteins are packed in the bag filter, put into 1.8 liters of renaturation buffers (damping fluid composition: 50mmol/L Tris-HCl pH 8.0,10mmol/L NaCl, 10 mmol/L EDTA, 2mmol/L DTT, 1 mmol/L GSSG, 1 mmol/L reductive glutathione, 0.5 mmol/L PMSF), 4 ℃ of lower magnetic forces stir dialysis, changed liquid once in per 5 hours, changing liquid dialyses 4 times with 1.8 liters of PBS for 4 times afterwards, front twice 12 hours/time, rear twice 6 hours/time.Collect liquid in the bag filter, 10000 rev/mins, 4 ℃ centrifugal 10 minutes, discard precipitation, with supernatant with 0.45 micron membrane filtration; Then concentrated with the 20KD concentration tube, 3000 rev/mins, 4 ℃ centrifugal 30 minutes, collect protein concentrate ,-20 ℃ frozen, for the preparation of detecting trace.

(2) swine foot-and-mouth disease virus nonstructural protein 3A BC Expression in Escherichia coli and purifying

The recombinant plasmid pGEM-T-3ABC that will contain the 3ABC gene cuts digestion with SalI and BglII enzyme, reclaims genes of interest fragment 3ABC.With XholI and BglII expression vector pTriEx-4Neo is digested, make it produce two cohesive ends identical with the purpose fragment; Connect two cohesive ends with T4 DNA Ligase, transform coli strain, 30 microlitre recombinant bacteriums are inoculated in 30 milliliters of LB nutrient culture media (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, carbenicillin 50mg/L) that contain carbenicillin, and 37 ℃, 250 rev/mins are shaken to OD600 and reach 0.5; Add isopropyl-β-D-sulfo-galactopyranoside derivant, making its final concentration is 0.5mmol/L, and in 37 ℃ of abduction deliverings 6 hours, it was centrifugal to express bacterium liquid, gathers in the crops bacterial precipitation.

Thalline is resuspended in 20 milliliters of damping fluids after (damping fluid composition: 10 mmol/L Tris-HCl pH, 8.0,150 mmol/L NaCl, 10 mmol/L EDTA, 10 % glycerine, 2 mmol/L DTT, 0.5 mmol/L PMSF) ultrasonication, centrifugal collecting precipitation is with washing lotion washing inclusion body 5 times (washing lotion composition: the Triton-100 of 1 % volume, 50 mmol/L Tris-HCl pH, 8.0,100 mmol/L NaCl, 10 mmol/L EDTA, 2 mmol/L DTT); Then will be deposited in 1 time (suspending liquid composition: 50 mmol/L Tris-HCl pH, 8.0,100 mmol/L NaCl, 10 mmol/L EDTA, 2 mmol/L DTT) of washing in 20 milliliters of suspending liquid, last centrifugation is the inclusion body behind the purifying.

It is in 8.0 the denaturant (denaturant composition: urea 8mol/L, NaCl 0.5mol/L, Tris-HCl 20mmol/L, EDTA 20mmol/L, 2% SDS) that the inclusion body of washing behind the purifying is dissolved in pH, in 4 ℃ of stir abouts 10 hours, the dissolving sex change, centrifugal removal insolubles obtains metaprotein; 90 milliliters of metaproteins are packed in the bag filter, put into 1.8 liters of renaturation buffers (damping fluid composition: 50mmol/L Tris-HCl pH 8.0,10mmol/L NaCl, 10 mmol/L EDTA, 2mmol/L DTT, 1 mmol/L GSSG, 1 mmol/L reductive glutathione, 0.5 mmol/L PMSF), 4 ℃ of lower magnetic forces stir dialysis, changed liquid once in per 5 hours, changing liquid dialyses 4 times with 1.8 liters of PBS for 4 times afterwards, front twice 12 hours/time, rear twice 6 hours/time.Collect liquid in the bag filter, 10000 rev/mins, 4 ℃ centrifugal 10 minutes, discard precipitation, with supernatant with 0.45 micron membrane filtration; Concentrated with the 20KD concentration tube, 3000 rev/mins, 4 ℃ centrifugal 30 minutes, collect protein concentrate ,-20 ℃ frozen, for the preparation of detecting trace.

3, the preparation of gold mark SPA glass wool:

Prepare aurosol with the sodium citrate reducing process: in 0.01～0.05% aqueous solution of chloraurate of 50～100 milliliters of boilings, add 2～4 milliliter 0.5～2% citric acid three sodium solution, obtain the collaurum about diameter 15 nanometers; K with 0.1mol/L ₂CO ₃Transfer collaurum pH to 8.5～9.5, mark ratio with 1:1000～1300 adds SPA to be marked in the aurosol of pH8.5～9.5, mark add after 10 minutes 20% PEG10000 to its ultimate density be 0.05%, 4 ℃, 1500～3000 rev/mins are centrifugal 20 minutes, remove unconjugated colloid gold particle, 4 ℃, 15000 rev/mins centrifugal 1 hour, abandon supernatant, obtain the SPA of colloid gold label; The colloid gold label SPA of 1:100～500 dilutions is adsorbed in the processed glass cotton, and 4 ℃ of low-temperature vacuum dryings are prepared gold mark SPA glass wool.

4, the detection principle of the utility model test strips:

The test strips test lead is inserted test serum, serum to be checked spreads to the cellulose rete together by the gold mark SPA that siphon drives in antibody to be checked and the gold mark SPA glass wool, the final absorbent material layer that infiltrates handle end, antibody to be checked combines with gold mark SPA in the diffusion process, the former detection trace of this bond and then the expression on cellulose membrane is combined, thereby demonstrates henna detection trace P and/or J; And the IgG antibody of the anti-SPA of sheep (rabbit) can be marked SPA with gold and is combined, and forms brownish red contrast trace C.If do not have FMDV antiviral antibody and vaccine antibody in the serum to be checked, test strips only shows a brownish red contrast trace C; If contain anti-FMDV antiviral antibody and/or vaccine antibody in the serum, then be combined with its gold mark SPA respectively, the detection trace P of the corresponding antigens on cellulose membrane and/or J are combined again, show that brownish red detects trace, positive mark; If show without any the brownish red trace on the cellulose membrane, show that then test strips had lost efficacy or misoperation.

5, the detection method of the utility model test strips:

(1) preparation of test sample: the aseptic blood of getting tested animal, and separation of serum, make 1:200 dilute serum doubly with physiological saline, obtain testing sample.

(2) detecting step: the test strips test lead is inserted in the serum to be detected, and insertion depth is no more than mark line, takes out test strips after about 30 seconds, and horizontal positioned 1～5 minute is observed testing result.

(3) result judges: if only show a brownish red contrast trace C on test strips cellulose rete, the expression testing result is negative, explanation does not detect FMDV antiviral antibody and FMDV vaccine antibody in tested serum, namely tested animal is without FMDV virus infections and FMDV vaccine immunity; If occur henna contrast trace C on the cellulose rete on the test strips and detect trace P, the expression testing result is positive, and namely detects the FMDV vaccine antibody in serum to be checked, and namely tested animal has carried out the FMDV vaccine immunity; Occur simultaneously if detect trace P, J, be illustrated in and detect FMDV virus infections antibody in the serum to be checked, namely tested animal has infected or has carried FMDV virus; If show without any the brownish red trace on the cellulose membrane band, show that then test strips had lost efficacy or operates wrong.

Following examples are used for further specifying the structure of the utility model test strips.

One: one step of embodiment is differentiated the test strips of mouth disease virus infection and vaccine immunity animal, referring to Fig. 1, Fig. 2, supporting layer 1 usefulness hard plastic strip of foil is made among the figure, sample adsorbing fiber layer 2 usefulness glass wool are made, the absorption of gold labeling antibody fibrage 3 usefulness has the glass wool of colloid gold label SPA to make, cellulose rete 4 adopts nitrocellulose filter to make, and absorbent material layer 5 usefulness absorbent filters are made; Sample adsorbing fiber layer 2, golden labeling antibody fibrage 3, cellulose rete 4, absorbent material layer 5 are sticked on the hard plastic strip of foil of supporting layer 1 successively the each other fiber of the intersection infiltration that crosses one another to left from right (test lead); The detection trace that the swine foot-and-mouth disease virus structural proteins VP1 of useful escherichia expression system expression and purifying and two kinds of antigen liquids of nonstructural protein 3A BC are printed on the cellulose rete 4 is respectively 6-P and 6-J, 7 is the contrast trace of printing with the IgG solution of the anti-SPA of sheep (or rabbit), and the combination trace band that detects trace, the formation of contrast trace is " || | "

8-1 covers sample adsorbing fiber layer 2 and the white diaphragm above the golden labeling antibody fibrage 3; be partial to sample adsorbing fiber layer 2 one side 0.5cm place in the corresponding diaphragm 8-1 of two-layer intersection position and be printed on mark line 9; the right-hand member of mark line 9 is printed on arrow and max printed words, absorbent material layer 5(handle end) on be coated with the diaphragm 8-2 of other color (such as yellow).

Embodiment two: test strips structure and embodiment one are basic identical, and difference is:

The absorption of gold labeling antibody fibrage 3 usefulness has the glass wool of colloid gold label SPA to make, express the also detection trace 6-P of the swine foot-and-mouth disease virus structural proteins VP1 antigen liquid printing of purifying with escherichia expression system, with the contrast trace C that the IgG solution of the anti-SPA of sheep (rabbit) is printed, two kinds of traces are arranged the combination trace band 7 that forms and are " || "

Embodiment three: test strips structure and embodiment two are basic identical, and difference is:

The absorption of gold labeling antibody fibrage 3 usefulness has the glass wool of colloid gold label SPA to make, express the also detection trace 6-J of the swine foot-and-mouth disease virus nonstructural protein 3A BC antigen liquid printing of purifying with escherichia expression system, 7 contrast traces for printing with the IgG solution of sheep (rabbit) anti-SPA, detect trace and contrast trace assembled arrangement and be " ● ● ".

Embodiment four: test strips structure and embodiment one are basic identical, and difference is:

Supporting layer 1 adopts the cardboard bar that does not absorb water to make, and sample adsorbing fiber layer 2 adopts nylon fiber to make, and cellulose rete 4 adopts cellulose membrane to make, and detection trace and contrast trace are arranged in parallel and are combined as " \ \ \ "

Embodiment five: test strips structure and embodiment one are basic identical, and difference is:

Supporting layer 1 adopts the cardboard bar that does not absorb water to make, and sample adsorbing fiber layer 2 adopts dacron to make, and cellulose rete 4 adopts the carboxymethyl cellulose films to make, detect trace and contrast trace be arranged in parallel and be combined as " ● ● ● ".

Embodiment six: test strips structure and embodiment one are basic identical, and difference is:

Supporting layer 1 adopts the cardboard bar that does not absorb water to make, and sample adsorbing fiber layer 2 adopts the nylon fiber film to make, and cellulose rete 4 adopts polyvinylidene fluoride (PVDF) cellulose membrane to make.

Embodiment seven: test strips structure and embodiment one are basic identical, and difference is:

Supporting layer 1 adopts the cardboard bar that does not absorb water to make, and sample adsorbing fiber layer 2 adopts nylon fibers to make, and cellulose rete 4 adopts cellulose membranes to make, detect trace and contrast trace be arranged in parallel and be combined as " ///".

Embodiment eight: test strips structure and embodiment one are basic identical, and difference is:

Supporting layer 1 adopts the cardboard bar that does not absorb water to make, and sample adsorbing fiber layer 2 adopts polyester cellulose to make, and cellulose rete 4 adopts the carboxymethyl cellulose film to make, and detection trace and contrast trace are arranged in parallel and are combined as " 10 ".

Embodiment nine: test strips structure and embodiment one are basic identical, and difference is:

Supporting layer 1 adopts the cardboard bar that does not absorb water to make, and sample adsorbing fiber layer 2 adopts polyester cellulose to make, and cellulose rete 4 adopts the polyvinylidene fluoride cellulose membrane to make, and detection trace and contrast trace are arranged in parallel and are combined as " ┬ ┬ ┬ ".

Embodiment ten: test strips structure and embodiment one are basic identical, and difference is:

Supporting layer 1 adopts the cardboard bar that does not absorb water to make, and sample adsorbing fiber layer 2 adopts the polyester cellulose film to make, and cellulose rete 4 adopts cellulose membrane to make, and detection trace and contrast trace are arranged in parallel and are combined as " ├ ├ ├ " or " ┤ ┤ ┤ ".

Embodiment 11: the susceptibility of the utility model test strips and specific test

Susceptibility: get after O type FMDV polypeptide vaccine and the inactivated vaccine immunity the 30th day each 1 part of pig serum, be respectively 1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800,1:25600,1:51200,1:102400 doubly dilute, totally 10 dilutabilitys, get each dilution serum 150 μ l, test strips with embodiment one is measured, measurement result after 5 minutes is read the bar instrument value of reading with TSR3000, estimates the susceptibility of test strips.The result shows, test strips to O type FMDV polypeptide vaccine and the immunity of polypeptide seedling after the sensitivity of the 30th day pig serum all can reach 1:12800.

Specificity: the equal no cross reactions of Positive Sera such as this test strips and swine fever, the B-mode brain of pig, pig blue-ear disease, porcine pseudorabies, pig annulus, specificity is 100%.

Claims (5)

1. a step is differentiated the test strips of mouth disease virus infection and vaccine immunity animal, contain supporting layer and adsorbed layer, adsorbed layer is attached on the supporting layer, adsorbed layer is followed successively by sample adsorbing fiber layer from test lead, gold labeling antibody fibrage, the absorbent material layer of cellulose rete and handle end, be provided with detection trace and contrast trace at the cellulose rete, it is characterized in that: the staphylococcal protein A of colloid gold label is arranged is SPA in absorption on the described golden labeling antibody fibrage, described detection trace is expressed with escherichia expression system and the swine foot-and-mouth disease virus structural proteins VP1 antigen of purifying and one or both printings in the nonstructural protein 3A BC antigen, and the contrast trace is printed with the IgG antibody of sheep or the anti-SPA of rabbit.

2. test strips according to claim 1 is characterized in that: described supporting layer is made with the hard plastic bar or the cardboard bar that do not absorb water; Sample adsorbing fiber layer is made with glass wool, nylon fiber or dacron; Gold labeling antibody fibrage is made with glass wool.

3. test strips according to claim 1, it is characterized in that: described cellulose rete is made with nitrocellulose filter, cellulose membrane, carboxymethyl cellulose film or polyvinylidene fluoride PVDF cellulose membrane; Described absorbent material layer is made with thieving paper.

4. test strips according to claim 1 is characterized in that: contain one/on the described cellulose rete when detecting trace, the spread pattern that detects trace, contrast trace be " ||", in " 10 ", " ┬ ┬ ", " ┴ ┴ ", " ├ ├ ", " ┤ ┤ " and " ● ● " any; Contain two/on the cellulose rete when detecting trace, the spread pattern that detects trace, contrast trace be " || |", " 10 ", " ┬ ┬ ┬ ", " ┴ ┴ ┴ ", " ├ ├ ├ ", " ┤ ┤ ┤ " and " ● ● ● " in any.

5. each described test strips according to claim 1～4; it is characterized in that: be coated with diaphragm at described sample adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer, the diaphragm corresponding with golden labeling antibody fibrage intersection at sample adsorbing fiber layer is printed with the sample mark line.

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