CN104730261A - Colloidal gold test strip for detecting chicken infectious anemia virus - Google Patents

Abstract

The invention discloses a colloidal gold test strip for detecting chicken infectious anemia virus. The colloidal gold test strip comprises a chicken anti-CIV (Huma Certain Immunodeficiency Virus) gold-labeled antibody glass fiber film, a nitrocellulose film, a water absorbing cushion and a PVC (polyvinyl chloride) bottom plate, wherein the nitrocellulose film is adhered to the center of the PVC bottom plate; the glass fiber film is formed by overlapping and connecting a sample cushion and a combining cushion; the other end of the combining cushion is adhered to one end with a T line, of the nitrocellulose film; the water absorbing cushion is adhered to one end with a C line, of the nitrocellulose film. With the adoption of the test strip for detecting, the sample is simply dropwise added onto the sample cushion of the test strip, and the operation is fast, simple and convenient, can be performed by one step, and is free of any instrument; the test strip is suitable for on-site use.

Description

A kind of CAV detects colloidal gold strip

Technical field

The invention belongs to virus detection techniques field, be specifically related to a kind of CAV and detect colloidal gold strip.

Background technology

Chicken infectious anemia (CIA) be by CAV (CIV) cause with chick alpastic anemia, total lymphoid Telatrophy, the subdermal muscle a kind of immunosupress sexually transmitted disease for feature such as hemorrhage.CIA causes the death increase of chicken group, survival rate and body weight gains to reduce.Again because immune organ damage causes serious immunosupress, reduce the resistibility of chicken group, increase chicken group to the neurological susceptibility of various cause of disease, reduce chicken group to various conventional vaccine immunity inoculation effect, very large difficulty is brought to the control of other diseases, can scabies secondary infection be caused simultaneously, carry out very large loss to poultry industrial belt.CIA is from Yuasa in 1979 etc. since Japanese reported first, and Germany, Sweden, Britain etc. are separated to CIV in succession.China was separated to CIV first in 1992, in recent years, had increase trend in this disease of certain areas.

At present, the method for CIA is diagnosed to comprise: Virus Isolation, serodiagnosis (comprising virus neutralization tests (NV), indirect immunofluorescence assay (IFA), enzyme linked immunosorbent assay (ELISA)) and biological technique method diagnosis (comprising round pcr, Nucleic Acid Probe Technique) etc.Although Virus Isolation is the effective means of diagnosis CIA, advantage is high specificity, wastes time and energy, and easily incurs loss through delay the treatment of this disease; Although the serological diagnostic method such as immunofluorescence, ELASA has trace, special, advantage fast and accurately, more complete instrument and equipment and veteran technician is needed to operate and judged result, inapplicable for veterinary station of basic unit or chicken house etc.The diagnosis of PCR and nucleic acid probe more needs special instrument and medicine, and with high content of technology, is difficult to apply in basic unit.Therefore, a kind of simple, quick, sensitive, cheap in the urgent need to setting up, be suitable for the method for quick of the CAV of basic unit's application.Oneself have developed the ELISA quick detection kit of chicken infectious anemia virus antibody both at home and abroad, this kit detects chicken infectious anemia virus antibody in serum, but in the operating process with this diagnostic kit, some reagent need extemporaneous preparation, and the use of this kit also seems not too convenient.Chicken infectious anemia virus antibody spot immune colloidal gold filtration assay detection method was carried out in state's inner beam intelligence choosing, was the detection to chicken infectious anemia virus antibody in serum, but there is the danger of silver nitrate environmental pollution.Up to now, yet there are no the report about CAV colloidal gold fast detecting test paper strip method establishment.

Summary of the invention

The present invention is directed to the technological deficiency that prior art detects fast to CAV, aim to provide a kind of CAV and detect colloidal gold strip, provide a kind of simple, quick, cheap, do not need any instrument, be particularly suitable for the new method that basic unit's application detects CAV (CIV).Both may be used for the diagnosis of chicken infectious anemia (CIA), and also may be used for the quarantine of CIA, there is broad prospect of application, for the generation of effective control CIA and popular significant.

The present invention realizes especially by following technical scheme:

A kind of CAV detects colloidal gold strip, comprise chicken anti-CIV gold labeling antibody glass fibre membrane, nitrocellulose membrane, adsorptive pads and PVC base plate, wherein nitrocellulose membrane is pasted on PVC base plate central authorities, glass fibre membrane is made up of sample pad and pad, pad end is pasted onto one end that nitrocellulose membrane draws T line, and overlapping 0.2cm, one end that nitrocellulose membrane draws C line is pasted with adsorptive pads, and overlapping 0.2cm.

Described nitrocellulose filter is prepared by the following method: nitrocellulose filter is cut into 0.5cm × 2.5cm, with coating buffer, anti-for chicken CIV antibody dilution is become 2 μ g/cm ²in one end of nitrocellulose filter line as detection line T line, with coating buffer, anti-for rabbit chicken igg antibody is diluted to 1.5 μ g/cm in the line of the nitrocellulose filter other end as detection line C line, line-to-line is apart from 0.5cm, and room temperature places 15min, is adsorbed in completely after on film until antibody, move to confining liquid and close 30min, then use 0.01mol/L phosphate buffer (pH 7.4) rinsing three times, 2min/ time, be placed in 37 DEG C of baking ovens dry, sealing, 4 DEG C of preservations.The dilutability of described chicken anti-CIV antibody and the anti-chicken igg antibody of rabbit is 1: 2.

Described chicken anti-CIV gold labeling antibody glass fibre membrane is prepared by the following method: the golden labeling antibody dilution of golden labeling antibody stoste is carried out 1: 2 dilution, by 4 μ g/cm ²be sprayed at glass fibre membrane pad end, the area of spraying is 0.5cm × 0.5cm, and 37 DEG C of absorption 1h, are then immersed in confining liquid and close 0.2cm, dry in 37 DEG C of incubators.4 DEG C save backup.

Described confining liquid is: BSA3g, sucrose 2.5g, polysorbas20 100 μ L, PVP-K300.3g, and Sodium azide 0.01g, adds 0.01mol/L Tris-HCl damping fluid (pH8.5) liquid and dissolve and be settled to 100mL.

Described glass fibre membrane is obtained by following preprocess method: glass fibre membrane is cut into 0.5cm × 3.0cm, with the Tris-HCl damping fluid of pH8.5 by glass fibre membrane rinsing three times, 5min/ time, by glass fibre membrane 37 DEG C oven dry good for rinsing, close with the 0.01mol/L phosphate buffer (pH 7.4) containing 2%BSA, 2.5% sucrose, 1% polysorbas20,0.3%PVP-K30 and 0.02% Sodium azide, dry in 37 DEG C of incubators.

Chicken of the present invention anti-CIV gold labeling antibody is prepared by the following method:

1) get 20mL collaurum, use 0.1mol K ₂cO ₃regulate colloidal gold solution pH to 8.5, by centrifugal for colloidal gold solution 3500r/min 15min, abandon precipitation, under magnetic stirring, in colloidal gold solution, slowly add 221.076 μ g/mL chicken CIV antibody is 44.22ug/mL to final concentration, Keep agitation 30min;

2) 10%BSA bovine serum albumin(BSA) is added to final concentration 1%, stirring at room temperature 10min;

3) 10%PEG 20000 to final concentration 0.2%, stirring at room temperature 10min is added.

4) by golden labeling antibody solution 4 DEG C of centrifugal 20min of 3500rpm, supernatant 4 DEG C of centrifugal 1h of 12000rpm, precipitation is diluted to original volume with golden labeling antibody conserving liquid, repeated centrifugation 2 times, precipitating with golden labeling antibody conserving liquid dilution is 1/20 of original volume, with 0.45 μm of membrane filtration, put 4 DEG C of Refrigerator stores for subsequent use.

Described golden labeling antibody conserving liquid is: BSA 0.1g, PEG20000 0.05g, NaN ₃0.02g, dissolves with the phosphate buffer of 30mL 0.01M pH7.4 and is settled to 100mL.

Collaurum of the present invention prepares collaurum by conventional trisodium citrate reduction method, is specially: by chloric acid gold (HAuCl ₄) be dissolved in tri-distilled water the aqueous solution being mixed with 0.01%; Get the HAuCl of 0.01% ₄aqueous solution 100ml is heated to boil, and accurately adds 1% trisodium citrate (Na under stirring ₃c6H ₅o ₇2H ₂o) aqueous solution 2ml.Continue heating and boil 15min.Now can be observed flaxen aqueous solution of chloraurate to gray very soon after sodium citrate adds, continue and change into black, being stable into orange red subsequently gradually.Original volume is returned to tri-distilled water after being cooled to room temperature.Same method prepares three times.4 DEG C for subsequent use.

Beneficial effect of the present invention is: sample consumption is minimum, can be low to moderate 2-3 and drip; Easy, single stepping, without the need to any instrument, is very applicable to onsite application; Safe and reliable, there is no the participation of objectionable impurities (as radioactive isotope, o-phenylenediamine, silver nitrate etc.), environmental pollution can not be caused; Detection required time is short, and a few minutes can go out result; Due to antigen-antibody reaction specificity, so sensitivity, specificity are higher; Result is stablized, and color is not subject to the impact of time and illumination, and experimental result can be preserved for a long time; Testing cost is low, if batch production, cost only needs 5-8 unit.

Accompanying drawing explanation

Fig. 1 is test strips structural representation of the present invention;

Fig. 2 is the vertical view of test strips of the present invention;

Fig. 3 is the specific detection of embodiment of the present invention test strips;

Fig. 4 is that the sensitivity of embodiment of the present invention test strips detects;

Fig. 5 is the Detection of Stability of embodiment of the present invention test strips;

Fig. 6 is that embodiment of the present invention test strips is to the example detection infecting CIV chicken;

Fig. 7 is infected group pcr amplification testing result; M:DNAMaker DL2000; 1-5: the heart, liver, spleen, lung, marrow;

Fig. 8 is control group pcr amplification testing result; M:DNA Maker DL2000; 1,2: positive control; 3-7: the heart, liver, spleen, lung, marrow; 8: blank;

Indicate in accompanying drawing 1,2 and illustrate: 1 sample pad; 2 pads; 3 nitrocellulose membranes; 4 detection lines; 5 nature controlling lines; 6 adsorptive pads; 7PVC base plate.

Embodiment

Below in conjunction with embodiment, the present invention is described further, the following stated, only to preferred embodiment of the present invention, not do other forms of restriction to the present invention, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed to the Equivalent embodiments of equal change.Everyly do not depart from the present invention program's content, any simple modification done following examples according to technical spirit of the present invention or equivalent variations, all drop in protection scope of the present invention.

Embodiment 1

A kind of CAV detects colloidal gold strip to be prepared by the following method:

One, the preparation of the anti-CIV antibody of chicken, purifying, detection

1, the preparation of the anti-CIV antibody of chicken

Chicken CIV Attenuate vaccine (the German Luo Man animal health company limited) immunity 4 times of SPF chicken, blood sampling, prepares serum antibody.

2, the purifying of the anti-CIV serum antibody of chicken

Successively twice purifying is carried out to serum antibody with sad-saturated ammonium sulfate method and antigen purification antibody act.

Conventional sad-saturated ammonium sulfate method:

Get serum 3ml, add the acetate buffer solution 6mL of 0.06M pH4.8, stir, it is sad at room temperature to add 225 μ L while stirring, stirs 30min, 4 DEG C of standing 2h.By serum solution 4 DEG C of centrifugal 30min of 800rpm, get supernatant, adjust pH to 7.4 with 2M NaOH.In supernatant, add equal-volume saturated ammonium sulfate solution while stirring, now ammonium sulfate final concentration is 50%, after stirring 30min, and 4 DEG C of standing 2h.The centrifugal 30min of 8000rpm, gets precipitation, precipitation is dissolved in 5mL PBS solution.In precipitation suspension, add saturated ammonium sulfate solution while stirring, make its final concentration be 45%, stir 30min, 4 DEG C of standing 2h.The centrifugal 30min of 8000rpm, gets precipitation, precipitation is dissolved in 2ml PBS solution.Loaded by precipitation suspension in bag filter, 4 DEG C, with PBS solution dialysis, 8h changes liquid once, dialysis 2d.Detect with Nai Shi reagent, till extracellular fluid dialysis is formed without yellow.0.45 μm of frit, 4 DEG C of storages.

Antigen purification antibody act:

The little bar nitrocellulose membrane of clip 1mm mono-, is placed in 1.5mL microcentrifugal tube, is immersed in 5min in the chicken CIV Attenuate vaccine solution of 1mL 1mg/mL.Take out film, be placed on filter paper dry.Film is positioned over the microcentrifugal tube that 1mL Buffer A damping fluid is housed, gently concussion mixing 30min.Remove Buffer A, wash once with fresh Buffer A, film is immersed in 2h in the anti-CIV antibody of 1mL purifying chicken, on vortex mixer, shakes mixing gently.Remove the anti-CIV antibody liquid of chicken of purifying, wash film 4 times with PBS, each 5min.

Film is placed in new configuration 100mM Glycine-HCl buffer (pH 2.8) antibody elution liquid, mixing 2min is shaken in have gentle hands, washing lotion is transferred to rapidly to be equipped with in 100u L 1M Tris (pH 7.4) centrifuge tube to neutralize, and makes antibody renaturation.1st wash-out 400 μ L antibody elution liquid, the 2nd, 3 use 200 μ L.In the liquid eluted, add 100 μ L BufferA with stabilization of antibodies, and be distributed into 30 μ L and often manage ,-20 DEG C of preservations.

3, the detection of the anti-CIV antibody of purifying chicken

The content of Conventional UV spectrophotometry purified antibodies is 221.076 μ g/mL.It is 1: 12800 that conventional indirect elisa method mensuration antibody purification is tired.

Two, the preparation of collaurum and qualification

1, the preparation of collaurum

Conventional trisodium citrate reduction method prepares collaurum.By chloric acid gold (HAuCl ₄) be dissolved in tri-distilled water the aqueous solution being mixed with 0.01%; The HAuCl of 0.01% ₄aqueous solution 100ml is heated to boil, and accurately adds 1% trisodium citrate (Na under stirring ₃c ₆h ₅o ₇2H ₂o) aqueous solution 2ml.Continue heating and boil 15min.Now can be observed flaxen aqueous solution of chloraurate to gray very soon after sodium citrate adds, continue and change into black, being stable into orange red subsequently gradually.Original volume is returned to tri-distilled water after being cooled to room temperature.Same method prepares three times.4 DEG C for subsequent use.

2, the qualification of collaurum

1. ocular estimate: the color of observing colloid gold, transparency and with or without suspended particle, tentatively determine the quality of the collaurum obtained, the collaurum outward appearance of preparation is claret, bright in colour, sparkling and crystal-clear bright, has light belt as seen in face of daylight lamp.

2. uv-spectrogram scanning method: carry out length scanning analysis at the colloidal gold solution of 400-700nm UV, visible light optical range to preparation, obtain collaurum visible region absorption spectrum, record maximum absorption wavelength.Determine its particle diameter and uniformity coefficient thereof further.The collaurum of preparation, in the scanning of 400-700nm place uv-spectrogram, obtains scanning spectra; The maximum absorption band of the collaurum of preparation is 524nm, there is linear relationship in collaurum particle diameter its maximum absorption band and particle diameter when 10-70nm: Y=0.4271X+514.56, the collaurum mean grain size drawing preparation is 22.1nm, belongs to and is relatively applicable to and protein bound particle diameter.

Three, the determination of the Optimal pH that is combined with collaurum of the anti-CIV antibody of chicken and optium concentration

1. gradient ocular estimate

Use 0.1mol/L K ₂cO ₃1mL colloidal gold solution in 10 test tubes is adjusted to different pH (6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10., 10.5) respectively, and often pipe adds the antibody liquid 0.1mL of 221.076 μ g/mL respectively, and after concussion 20min, room temperature places 10min; Then often pipe adds the 10%NaCl solution of 0.1mL respectively, and after concussion mixing 10min, ambient temperatare puts 2h; The change of observing colloid gold color, record keeps red minimum pH, is Optimal pH.The test tube of result collaurum pH8.5-10.5 all keeps red, so optimal pH is 8.5.

2. OD value curve method

The 10%NaCl solution of 0.1mL is respectively added in 10 test tubes of above-mentioned ocular estimate, after after concussion mixing 10min, ambient temperatare puts 2h, the centrifugal 30min of 3500rpm, by the OD value of spectrophotometric determination solution at 520nm place, with OD value for ordinate, pH value is that horizontal ordinate makes a curve, and getting the point that curve is close with transverse axis is at first optimum pH.Result optimum pH is 8.5, illustrate when collaurum pH is 8.5 collaurum and CIV antibody in conjunction with best.

2, the mensuration of optium concentration that is combined with collaurum of CIV antibody

1. gradient ocular estimate

Colloidal gold solution is transferred to pH 8.5,10 test tubes are often managed and are added 1mL, with PBS damping fluid, CIV antibody stoste is pressed 1: 2,1: 4, doubling dilution becomes variable concentrations, 2 ~ No. 10 test tubes add the anti-CIV antibody of variable concentrations chicken respectively and add 1mL, and No. 1 pipe adds tri-distilled water 1mL, and after concussion mixing 20min, ambient temperatare puts 10min.Then No. 1-9 pipe respectively adds 0.1mL 10%NaCl, and No. 10 pipes add the tri-distilled water of 0.1mL, and after concussion mixing 10min, ambient temperatare puts 10min.Observe the change of each test tube color.The anti-CIV antibody concentration of chicken is 110.54,55.27, and the test tube collaurum color of 36.85 μ g/mL keeps basic redness constant, and minimum antibody consumption is 36.85 μ g/mL.Increase by the antibody consumption of 20% on this basis again, the optium concentration that CIV antibody is combined with collaurum is 44.22 μ g/mL.

2. OD value curve method

Respectively add the dilution of 2mL tri-distilled water in the test tube of gradient ocular estimate after, absorbance is measured at wavelength 520nm place, with OD value for ordinate, the anti-CIV antibody concentration of chicken is horizontal ordinate curve plotting, the minimum stable antibody concentration of that antibody concentration located that curve is close with transverse axis at first needed for stable 1mL collaurum, increase by the antibody consumption of 20% on this basis again, be the optium concentration that CIV antibody is combined with collaurum.Show that minimum antibody stable quantity is 36.85ug/mL, the optium concentration that the anti-CIV antibody of chicken is combined with collaurum is 44.22 μ g/mL.

Four, the preparation of chicken anti-CIV gold labeling antibody, purifying

1. the preparation of chicken anti-CIV gold labeling antibody

1. get 20mL collaurum, use 0.1mol K ₂cO ₃regulate colloidal gold solution pH to 8.5, by centrifugal for colloidal gold solution 3500r/min 15min, abandon precipitation.Under magnetic force speed stirs, in colloidal gold solution, slowly add 221.076 μ g/mLCIV antibody to final concentrations is 44.22ug/mL Keep agitation 30min.

2. 10%BSA bovine serum albumin(BSA) is added to final concentration 1%, stirring at room temperature 10min.

3. 10%PEG 20000 to final concentration 0.2%, stirring at room temperature 10min is added.

2. the purifying of chicken anti-CIV gold labeling antibody

Adopt low temperature supercentrifugation purifying chicken anti-CIV gold labeling antibody, to remove the polymkeric substance formed in unlabelled chicken anti-CIV antibody, unlabelled collaurum and labeling process.

By golden labeling antibody solution 4 DEG C of centrifugal 20min of 3500rpm, discard precipitation; Supernatant 4 DEG C of centrifugal 1h of 12000rpm, abandoning supernatant, precipitates and is diluted to original volume with golden labeling antibody conserving liquid, repeated centrifugation 2 times, and precipitating with golden labeling antibody conserving liquid dilution is 1/20 of original volume, with 0.45 μm of membrane filtration, puts 4 DEG C of Refrigerator stores for subsequent use.

Gold labeling antibody conserving liquid compound method: BSA 0.1g, PEG200000.05g, NaN ₃0.02g, dissolves with the phosphate buffer of 30mL 0.01M pH7.4 and is settled to 100mL, and 4 DEG C save backup;

3, the Quality Identification of chicken anti-CIV gold labeling antibody

Get rabbit anti-chicken antibody point sample on NC film, the effect of direct and chicken anti-CIV gold labeling antibody, has obvious punctation as seen, shows that chicken anti-CIV gold labeling antibody has activity.

The preparation of five test strips ingredients

1, the process of glass fibre membrane

Import glass fibre membrane (Ahlstrom8964) is cut into 0.5cm × 3.0cm, with the Tris-HCl damping fluid of pH8.5 by glass fibre membrane rinsing three times, and 5min/ time, by glass fibre membrane 37 DEG C oven dry good for rinsing.Close with the 0.01mol/L phosphate buffer (pH 7.4) containing 2%BSA, 2.5% sucrose, 1% polysorbas20,0.3%PVP-K30 and 0.02% Sodium azide, dry in 37 DEG C of incubators.

The preparation of 2 chicken anti-CIV gold labeling antibody glass fibre membrane

The golden labeling antibody dilution of golden labeling antibody stoste is carried out 1: 2 dilution, by 4 μ g/cm ²be sprayed at (0.5cm × 0.5cm) on glass fibre membrane one end, 37 DEG C of absorption 1h, are then immersed in confining liquid and close 0.2cm, dry in 37 DEG C of incubators.4 DEG C save backup.

The preparation of confining liquid: BSA3g, sucrose 2.5g, polysorbas20 100 μ L, PVP-K300.3g, Sodium azide 0.01g, adds 0.01mol/L Tris-HCl damping fluid (pH8.5) liquid and dissolves and be settled to 100mL

3, on cellulose nitrate (NC) film, coated antibody is dilution determines

Anti-for chicken CIV antibody and the anti-chicken igg antibody of rabbit are done 1: 2 respectively, 1: 4,1: 6,1: 8,1: 10 dilution, point sample 2 μ g/cm on NC film ²be soaked in after drying in golden labeling antibody solution, take out after 2min, with the PBS buffer solution three times of 0.01M pH7.4, every minor tick 3min, the colour developing situation of observation point sampling point.1: 2 antibody dilution colour developing is best as a result.

4 antibody bags are by nitrocellulose filter

Millipore 135 model nitrocellulose filter is cut into 0.5cm × 2.5cm, with coating buffer, anti-for chicken CIV antibody dilution is become 2 μ g/cm ²in one end of NC film line as detection line T line, with coating buffer, anti-for rabbit chicken igg antibody is diluted to 1.5 μ g/cm ²in the line of the NC film other end as detection line C line, line-to-line is apart from 0.5cm, room temperature places 15min, be adsorbed in completely after on film until antibody, move to confining liquid and close 30min, then use 0.01mol/L phosphate buffer (pH 7.4) rinsing three times, 2min/ time, be placed in 37 DEG C of baking ovens dry, sealing, 4 DEG C of preservations;

The assembling of six test strips

As depicted in figs. 1 and 2, PVC base plate is cut into 2.5cm wide/bar, its central authorities of flat stickup are stretched by drawing the NC film having detection line and nature controlling line, glass fibre membrane (pad and sample pad) is pasted onto one end that NC film draws T line, and overlapping NC film end 0.2cm, upper adsorptive pads is pasted in one end that NC film draws C line, and overlapping NC film end 0.2cm, is the test strips assembled.After the test strips assembled is flattened, be cut into 0.5cm/ bar with scissors, hermetically drying, 4 DEG C of preservations.

The Cleaning Principle of test paper of the present invention is: by test strips sample pad dripping tri-distilled water 2, act on 10min under room temperature, only occurs a red line at nature controlling line C line, and detection line T line is not aobvious red, shows that negative findings is set up.Test strips sample pad will drip the sample of 2 known CIV, acting on 10min under room temperature, all there is red line in detection line T line and nature controlling line C line.To be added in sample pad containing CIV testing sample and by capillary action upwards swimming, first there is specific immune response with the anti-CIV of the chicken on pad gold labeling antibody in CIV, the compound formed is captured when moving forward to the anti-CIV antibody of chicken under capillary action, namely there is double antibodies sandwich specific bond, at detection line place, colloid gold particle enrichment takes on a red color, the golden labeling antibody do not combined continues swimming forward, with be fixed on the anti-chicken igg antibody of the rabbit on NC film and be combined and form compound, golden labeling antibody presents redness in the enrichment of nature controlling line place.Negative testing sample to be added in sample pad and by capillary action upwards swimming, at detection line, anti-CIV gold labeling antibody does not react with chicken, swimming is to nature controlling line forward for chicken anti-CIV gold labeling antibody, and chicken anti-CIV gold labeling antibody and the anti-chicken igg antibody of rabbit form compound, and colloid gold particle enrichment takes on a red color.

Embodiment 2 ELISA test strip

1) specific test of test strips

Test strips sample pad drips CIV, AIV, NDV, MDV virus liquid 2 respectively, observes after 10min, as shown in Figure 3, detection line and the nature controlling line of the test strips of result dropping CIV virus liquid all show redness; The test strips dripping AIV, NDV, MDV virus liquid is all that only nature controlling line display is red, and detection line is not aobvious red.Illustrate that this colloidal gold strip with AIV, NDV, MDV virus, cross reaction does not occur.

2) the sensitivity test of test strips

Test strips sample pad drips respectively containing 10, the virus liquid 2 of the CIV of 1,0.1,0.01 μ g/mL, observe after 10min, as shown in Figure 4, during the CIV of result display 10,1 μ g/mL, detection line colour developing is good, during the CIV of 0.1 μ g/mL, detection line still has colour developing, but be not too clear, during the CIV of 0.01 μ g/mL, detection line does not develop the color, and illustrates that test strips is compared with susceptibility.

3) stability test of test strips

By test strips polybag and foil sealing, add drying agent, respectively at 4 DEG C, 25 DEG C placements, 10 are extracted out at random every three months, detect once with the CIV liquid of 1 μ g/mL, as shown in Figure 5, result display was placed in 4 DEG C of test strips of placing 12 months time, and the color of the colour developing band of detection line is still very clear; 25 DEG C of colors of placing the colour developing band of 3 months, 6 months and 9 months detection lines are all very clear, and when 12 months, detection line is not aobvious red.Illustrate that test strips at least can preserve 12 months at 4 DEG C, at least can preserve 9 months at 25 DEG C.The stability of this test strips meets the index of national defined.

4) replica test of test strips

Get 15 test strips of same batch of preserving at 25 DEG C, detect once every day with the CIV liquid of 1 μ g/mL, continuous detecting 5 days.Get 20 in the test strips of 25 DEG C of different batches preserved, detect with the CIV liquid of 1 μ g/mL, detect 5 batches.The repeatability of test strips is determined by the colour developing change observing test strips.Result is as shown in table 1, and the coefficient of variation repeated in batch is 3.7%, and the coefficient of variation is less than 10%.The coefficient of variation repeated between batch is 4.61%, and the coefficient of variation is less than 15%.Illustrate that this test strips has good repeatability, the repeatability of this test paper of same batch can reach 97%; The repeatability of the test strips of different batches also reaches 97%, and false positive rate and false negative rate are 0.

The repeatability of table 1 test strips

Embodiment 3 CAV detects the application of colloidal gold strip

1, artificial infection CIV prepares disease chicken model

(Anhui Science and Technology College's poultry diease control and prevention of disease laboratory is separated to have pathogenic chicken infectious anemia virus, called after Anhui-2011-1) in liquid and EP pipe, 30min is processed in 70 DEG C of water-baths, then 50% ether hybrid processing is added 1 hour, by the subcutaneous vaccination for the treatment of fluid neck in 1 age in days SPF chick, every 0.5mL, 1 age in days SPF chicken 8 is infected in hypodermic injection, simultaneously with not injecting 8 chickens in contrast.Infectable infection, after 14 days, occurs that cockscomb is pale, hypoevolutism, spirit depressed, become thin, wing dermatitis, there are the symptoms such as petechial hemorrhage at limb wing place.Dissect and find that blood is thin, cruor time extending, marrow change most feature, femoral marrow is fat-like, yellow or faint yellow.Thymus gland and bursa of farbricius atrophy or diminish.Possesses the Clinical symptoms of chicken infectious anemia.Blank group well-grown.Asepticly take that disease heart is dirty, liver, spleen, lungs and marrow.

2, ELISA test strip

Get the little scissors of a fritter liver to shred, add about 1ml damping fluid 1.Test strips sample pad drips disease chicken liver sample 2, observes after 10min, as shown in Figure 6, detection line and the nature controlling line of 8 infected group chicken test strips all show redness to result, and liver pathological material of disease tests positive is described.Only nature controlling line display is red for 8 control group chicken test strips, illustrates that liver pathological material of disease detects and is negative.

3, PCR method Isolation and ldentification chicken infectious anemia virus

1) extraction of the pathological material of disease genomic DNA gathered:

1. get a fritter heart respectively, liver, spleen, lung, marrow is placed in 1.5Ep pipe, shred with little scissors, as well careful.Add about 1ml damping fluid 1, with pipettor mixing, the centrifugal 5min of 10000rpm, abandons supernatant.Repeat aforesaid operations twice.If impure, aforesaid operations can be continued, until sediment is in white or red and white.

2. the ddH of 300-400ul is added ₂o mixes cell.

3.-20 DEG C of multigelation cells 3 times, each 30min.

4. by the centrifugal 1min of cell 5000rpm after freeze thawing, get supernatant and add in EP pipe.

5. add isopyknic Tris balance phenol, fully mix, the centrifugal 10min of 12000rpm, gets supernatant.

6. add isopyknic Tris balance phenol and chloroform (1: 1), fully mix, the centrifugal 10min of 12000rpm, gets supernatant.

7. add isopyknic chloroform, fully mix, the centrifugal 10min of 12000rpm, gets supernatant.

8. add the freezing absolute ethyl alcohol of two volumes, mix gently, in-20 DEG C of precipitation 30min, then the centrifugal 5min of 12000rpm, pours liquid.

9. 70% ethanol rinse twice (adding along edge), dries.

10. 30ulTE is deposited in 4 DEG C of preservations.

2) qualification of isolated viral

The extract of system to the marrow of infected group and control group chicken carries out pcr amplification routinely, and PCR reaction system is 20ul, wherein

Reaction conditions: 94 DEG C of 5min, 95 DEG C of 50s, 57 DEG C of 50s, 72 DEG C of 1min, 72 DEG C of 10min, 30cycles.

As shown in Figure 7, the amplified production after infected group pcr amplification testing result shows its electrophoresis conforms to object clip size, has infection activity.As shown in Figure 8, control group PCR result is feminine gender, to show in non-infected group body not this cause of disease.

The monitoring result of this test strips is consistent with PCR method result, but test strips monitoring has only used 5min, PCR method to use 5 ~ 6 hours, and easily pollutes in the leaching process of DNA.

Claims (7)

1. a CAV detects colloidal gold strip, comprise chicken anti-CIV gold labeling antibody glass fibre membrane, nitrocellulose membrane, adsorptive pads and PVC base plate, it is characterized in that: described nitrocellulose membrane sticks in PVC base plate central authorities, glass fibre membrane was made up of connecting overlapping with pad one end of sample pad, the other end of pad be pasted onto one end that nitrocellulose membrane draws T line, one end that nitrocellulose membrane draws C line is pasted with adsorptive pads.

2. a kind of CAV according to claim 1 detects colloidal gold strip, it is characterized in that: described nitrocellulose filter is prepared by the following method: with coating buffer, anti-for chicken CIV antibody dilution is become 2 μ g/cm in one end of nitrocellulose filter line as detection line T line, with coating buffer, anti-for rabbit chicken igg antibody is diluted to 1.5 μ g/cm in the line of the nitrocellulose filter other end as detection line C line, line-to-line is apart from 0.5cm, room temperature places 15min, be adsorbed in completely after on film until antibody, move to confining liquid and close 30min, then pH 7.4 is used, the phosphate buffer rinsing of 0.01mol/L three times, 2min/ time, be placed in 37 DEG C of baking ovens dry, sealing, 4 DEG C of preservations.

3. a kind of CAV according to claim 1 detects colloidal gold strip, it is characterized in that: described chicken anti-CIV gold labeling antibody glass fibre membrane is prepared by the following method: the golden labeling antibody dilution of golden labeling antibody stoste is carried out 1: 2 dilution, glass fibre membrane pad end is sprayed at by 4 μ g/cm2, spray area is 0.5cm × 0.5cm, 37 DEG C of absorption 1h, then be immersed in confining liquid and close 0.2cm, dry in 37 DEG C of incubators, 4 DEG C save backup.

4. a kind of CAV according to claim 3 detects colloidal gold strip, it is characterized in that: described confining liquid is: BSA3g, sucrose 2.5g, polysorbas20 100 μ L, PVP-K30 0.3g, Sodium azide 0.01g, adds 0.01mol/L Tris-HCl damping fluid (pH8.5) liquid and dissolves and be settled to 100mL.

5. a kind of CAV according to claim 3 detects colloidal gold strip, it is characterized in that: described glass fibre membrane is obtained by following preprocess method: the Tris-HCl damping fluid of glass fibre membrane pH8.5 is by glass fibre membrane rinsing three times, 5min/ time, by glass fibre membrane 37 DEG C oven dry good for rinsing, close with the phosphate buffer of 0.01mol/L, the pH 7.4 containing 2%BSA, 2.5% sucrose, 1% polysorbas20,0.3%PVP-K30 and 0.02% Sodium azide, dry in 37 DEG C of incubators.

6. the preparation method of the anti-CIV gold of chicken described in claim 3 labeling antibody, is characterized in that: comprise the following steps:

1) get 20mL collaurum, use 0.1mol K ₂cO ₃regulate colloidal gold solution pH to 8.5, by centrifugal for colloidal gold solution 3500r/min 15min, abandon precipitation, under magnetic force speed stirs, in colloidal gold solution, slowly add 221.076 μ g/mLCIV antibody to final concentrations is 44.22ug/mL Keep agitation 30min;

2) 10%BSA bovine serum albumin(BSA) is added to final concentration 1%, stirring at room temperature 10min;

3) 10%PEG 20000 to final concentration 0.2%, stirring at room temperature 10min is added;

4) by golden labeling antibody solution 4 DEG C of centrifugal 20min of 3500rpm, supernatant 4 DEG C of centrifugal 1h of 12000rpm, precipitation is diluted to original volume with golden labeling antibody conserving liquid, repeated centrifugation 2 times, precipitating with golden labeling antibody conserving liquid dilution is 1/20 of original volume, with 0.45 μm of membrane filtration, put 4 DEG C of Refrigerator stores for subsequent use.

7. preparation method according to claim 6, is characterized in that: described conserving liquid is: BSA 0.1g, PEG20000 0.05g, NaN ₃0.02g, dissolves with the phosphate buffer of 30mL 0.01M pH7.4 and is settled to 100mL.