CN102879573B - Immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing immune colloidal gold test paper - Google Patents
Immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing immune colloidal gold test paper Download PDFInfo
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- CN102879573B CN102879573B CN201210379154.7A CN201210379154A CN102879573B CN 102879573 B CN102879573 B CN 102879573B CN 201210379154 A CN201210379154 A CN 201210379154A CN 102879573 B CN102879573 B CN 102879573B
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Abstract
The invention discloses immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing the immune colloidal gold test paper. The test paper consists of a test paper box and a test paper tape, and the test paper tape is made by sequentially bonding a hard polyvinyl chloride lining plate, a nitrocellulose membrane, a long colloidal gold combination pad, a short colloidal gold combination pad, a sample pad and an absorption pad. VP2 gene protein monoclonal antibody- colloidal gold biomarkers resistant to mink-source canine distemper viruses and parvoviruses are sprayed on the long and short colloidal gold combination pads respectively. Two detection lines formed by rabbit anti-mink source canine distemper virus and parvovirus antibodies and a quality control line formed by a rabbit antimouse antibody are sprayed on the nitrocellulose membrane. The test paper is applicable to quick detection of pestilences of foxes, raccoon dogs and minks, and detection of two pestilences including canine distemper and canine parvo of foxes, raccoon dogs and minks can be simultaneously completed by the same test paper, and detection is simple, convenient, quick and accurate.
Description
Technical field
The present invention relates to the detection utensil of cultivated animals epidemic disease, be specifically related to a kind ofly to examine fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus simultaneously.The invention still further relates to the preparation method of this test paper.
Background technology
Canine distemper (Canine distemper, CD) is a kind of high degree in contact sexually transmitted disease that in the dog and carnivorous order caused by CDV (Canine distemper virus, CDV), many animals comprise fox, recoon dog and mink; Parvovirus (Canine Parvo, CP) is a kind of acute infectious disease that the dog class animal caused by parvovirus (Canine Parvovirus, CPV) comprises fox, recoon dog and mink.These two kinds of infectious diseases often cause the animal morbidities such as large quantities of fox, recoon dog and mink, and case fatality rate is generally between 30% ~ 80%.Along with the increasing considerably of the number of animals raised of the fur-bearing animals such as China fox, recoon dog and mink, and strange land exchange increase, the incidence of disease of these two kinds of infectious diseases in the fur-bearing animals such as China fox, recoon dog and mink and fatal rate all have the trend of rising, be at present maximum epidemic disease is endangered to Fur Animal Feeding industry such as China fox, recoon dog and minks, receive and pay attention to widely.At present, the method such as virus purification, electron microscopy, neutralization test, enzyme linked immunosorbent assay (ELISA), PCR (PCR) has played effect in the diagnosis of canine distemper, parvovirus.But these methods respectively have drawback, Virus Isolation and the neutralization test process of routine are loaded down with trivial details, consuming time longer; Electron microscopy is difficult to apply in basic unit; Protocols in Molecular Biology is as responsive, special in PCR etc., and technology and equipment requires high, and these detection methods are difficult at veterinary clinic onsite application.
Immunochromatography colloidal gold technique has fast easy, high specificity, and susceptibility is high, naked eyes interpretation, and experimental result is easily preserved, and without the need to the advantage such as special instruments and equipment and reagent, has been widely used in veterinary clinic disease detection or diagnosis.At present, detect the colloid gold test paper that numerous epidemic diseases of dog have its corresponding epidemic disease, and be not exclusively used in the colloid gold test paper detecting fox, recoon dog and mink epidemic disease.If clinically fox, recoon dog and mink generation canine distemper or (with) parvovirus, can only use detect dog canine distemper or (with) parvovirus colloid gold test paper, and prepare dog canine distemper or (with) canine parvovirus prevention needed for parvovirus colloid gold test paper or (with) antigen of the monoclonal antibody of parvovirus is all separated from dog, be used on fox, recoon dog, mink and may cause false positive or false negative result, poor accuracy.So far, the monoclonal antibody that the pathogen be not also separated from mink is prepared as antigen makes immunochromatography colloid gold test paper, does not more have to detect fox, recoon dog, the canine distemper of mink and the test paper of parvovirus two kinds of epidemic diseases simultaneously.
Summary of the invention
The object of the invention is to for the problems referred to above, there is provided a kind of and can detect fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus simultaneously, this test paper is easy to use, simple to operate, detect fast, accurately, and fox, racoon dog, the canine distemper of ermine and parvovirus can be detected with same test paper simultaneously.The present invention also aims to provide a kind of preparation method that simultaneously can detect fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus.
The technical scheme realizing above-mentioned purpose is: a kind ofly can detect fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus simultaneously, the test strips cartridge of well and colour developing district opening is set above being included in, and is arranged on the test strips in this test strips cartridge, described test strips includes rigid polyvinyl chloride liner plate, nitrocellulose filter, sample pad and absorption pad, described rigid polyvinyl chloride liner plate is arranged on the box bottom surface of described test strips cartridge, described nitrocellulose filter is placed in above described rigid polyvinyl chloride liner plate, the long gold conjugation pad be placed in above described nitrocellulose filter and the short gold conjugation pad be placed in above described long gold conjugation pad is provided with in one end of described test strips cartridge, described sample pad is placed in above described short gold conjugation pad, corresponding with described well above this sample pad, the other end that described absorption pad is placed in described test strips cartridge is positioned at above described nitrocellulose filter, described long gold conjugation pad and short gold conjugation pad are coated with respectively the VP2 gene protein monoclonal antibody-colloid gold label thing of anti-ermine source CDV and parvovirus, the VP2 gene protein monoclonal antibody of this anti-ermine source CDV and parvovirus is the VP2 gene protein immunity Balb/c mouse of the parvovirus using the band of the ermine source CDV of purifying and the Yeast expression of restructuring histidine-tagged, through Fusion of Cells, the VP2 gene protein monoclonal antibody that screening obtains anti-ermine source CDV and parvovirus is that hybridoma cell line is secreted, described nitrocellulose filter is coated with respectively two detection lines of rabbit anti-ermine source CDV and parvovirus antibody formation and a nature controlling line of rabbit anti-mouse antibody formation, these two detection lines are corresponding with described colour developing district opening with nature controlling line.
The described preparation method that simultaneously can detect fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus, it comprises the following steps:
1, the VP2 gene protein of the histidine-tagged parvovirus of the band of the ermine source CDV of purifying and the Yeast expression of restructuring is prepared;
The VP2 gene protein immunity Balb/c mouse of the parvovirus 2, using the band of the ermine source CDV of purifying and the Yeast expression of restructuring histidine-tagged respectively, through Fusion of Cells, screening obtains the hybridoma cell line of the VP2 gene protein monoclonal antibody of secreting anti-ermine source CDV and parvovirus;
3, the VP2 gene protein monoclonal antibody of anti-ermine source CDV and parvovirus is prepared;
4, react with trisodium citrate and gold chloride and prepare collaurum;
5, the VP2 gene protein monoclonal antibody of anti-ermine source CDV obtained for step 3 and parvovirus is added respectively in the collaurum that step 4 prepares, obtain the VP2 gene protein monoclonal antibody-colloid gold label thing of anti-ermine source CDV and parvovirus respectively;
6, the VP2 gene protein monoclonal antibody-colloid gold label thing of anti-ermine source CDV and parvovirus is sprayed on long gold conjugation pad and short gold conjugation pad respectively;
7, rabbit anti-ermine source CDV and parvovirus antibody is prepared, preparation rabbit anti-mouse antibody;
8, the antibody of anti-for rabbit ermine source CDV and parvovirus is sprayed on formation two detection lines on nitrocellulose filter, and rabbit anti-mouse antibody is sprayed on a formation nature controlling line on nitrocellulose filter;
9, rigid polyvinyl chloride liner plate, nitrocellulose filter, long gold conjugation pad, short gold conjugation pad, sample pad, absorption pad are bonded together composition test strips in order, and load fixing on request for this test strips in test strips cartridge, obtain described to detect fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus simultaneously.
The immunochromatography reaction that the present invention develops the color by colloid gold label, fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus can be detected with preparation simultaneously, structure is more reasonable, whether application rabbit anti-ermine source CDV and parvovirus antibody, as detection line, are set up competition immunochromatography and are detected fast in testing sample containing CDV and parvovirus.
Compared with prior art, the present invention has following outstanding advantages:
1, be applicable to fox, racoon dog, the canine distemper of ermine and the quick detection of parvovirus, and CDV and parvovirus can be detected with same test paper simultaneously.
2, detection paper result high specificity of the present invention, highly sensitive.CDV or parvovirus cell median infective dose (TCID
50) reach 9.4 × 10
5be diluted to 1:512 still can detect.
3, test paper of the present invention does not need any instrument and equipment, easy to carry, and testing cost is low.
4, test paper of the present invention easy control simple to operate, operates without the need to professional.
5, examination bar of the present invention is preserved conveniently, good stability.
Accompanying drawing explanation
Accompanying drawing is the structural representation that the present invention can detect fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus simultaneously.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
As accompanying drawing, the present invention can detect fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus simultaneously and include test strips and test strips cartridge 3, and test strips is fixed in test strips cartridge 3.Test strips cartridge 3 has box body and upper cover two parts, offers well 1, offer colour developing district opening 2 at upper cover medium position in the keep left position of side of upper cover.Test strips is combined into one by bonding by rigid polyvinyl chloride liner plate 4, nitrocellulose filter 5, sample pad 6, absorption pad 7, long gold conjugation pad 8 and short gold conjugation pad 9 and is formed, its concrete unitized construction: nitrocellulose filter 5 is placed in above rigid polyvinyl chloride liner plate 4, the side that keeps left on nitrocellulose filter 5 sets gradually long gold conjugation pad 8, short gold conjugation pad 9 and sample pad 6 from the bottom to top, and absorption pad 7 is arranged on nitrocellulose filter 5 position on the right side above.Test strips can adopt bonding, that draw-in groove is fixed, box body upper cover compresses mode to be fixed in test strips cartridge.Two detection lines 10 and a nature controlling line 11 is coated with at the middle part of nitrocellulose filter 5.Long gold conjugation pad 8 and short gold conjugation pad 9 are made up of glass fibre element film, a long gold conjugation pad 8 and a short gold conjugation pad 9 are one group, this two gold conjugation pad sprays respectively CDV monoclonal antibody and the parvovirus VP2 gene protein monoclonal antibody of the water resistant ermine of colloid gold label, described anti-ermine source CDV and the VP2 gene protein monoclonal antibody of parvovirus are the VP2 gene protein immunity Balb/c mouse of the parvovirus using the band of the ermine source CDV of purifying and the Yeast expression of restructuring histidine-tagged, through Fusion of Cells, the VP2 gene protein monoclonal antibody that screening obtains anti-ermine source CDV and parvovirus is that hybridoma cell line is secreted.Nitrocellulose filter 5 is the detection film of test strips, the side that keeps left above be two detection lines 10, nature controlling line 11 be positioned at right side, two detection lines 10 spray rabbit anti-ermine source CDV and parvovirus antibody respectively, and nature controlling line 11 sprays rabbit anti-mouse antibody.When containing corresponding virus in test sample, virus can be distinguished and first marks anti-ermine source CDV monoclonal antibody with golden and parvovirus VP2 gene protein monoclonal antibody is combined, then the antibody under chromatography effect on corresponding detection line is combined and forms macroscopic colour band after enrichment, and then judges according to colour developing result.
The present invention can detect simultaneously fox, racoon dog, the canine distemper of ermine and the immunochromatography colloid gold test paper of parvovirus concrete preparation method be:
1, the preparation of anti-ermine source CDV monoclonal antibody: by the CDV Hebei strain culture purified be separated from mink, with CDV Hebei, the ermine source strain immunity Balb/c mouse of culture purified, 1st abdominal part hypodermic complete Freund's adjuvant+ermine source CDV antigen (1:1) 100 μ L, by the amount of a 50 μ g albumen/mouse, the Balb/c female mice of initial immunity 4 no-special pathogens (SPF).After one week subcutaneous first time booster immunization, injection incomplete Freund's adjuvant+CDV antigen (1:1) 100 μ L, antigen amount is 50 μ g; After 14d, lumbar injection incomplete Freund's adjuvant+ermine source CDV antigen (1:1) 200 μ L, antigen amount is 100 μ g, interval 14d, tail vein blood, measures immune effect.Above-mentioned, after antibody titer reaches requirement, before fusion, 3d (being separated by more than 2 weeks with immunity last time) uses non-emulsified antigen booster immunization once.After 3d, aseptic spleen of getting merges.Through Fusion of Cells, the antigen coated ELISA Plate of cell conditioned medium is screened, and establishes positive and negative to contrast simultaneously.The positive colony obtained is through repeated detection, and secretory antibody is stablized.After limiting dilution assay clones 3 times, positive rate reaches 100%.Be total to obtain 14 strain positive colony cells in accordance with the law, use the ermine source CDV coated elisa plate of purifying again, make compound detection, by the cell line of the 14 strain positives, again use ermine source CDV wrapper sheet, adopt enzyme-linked immunologic adsorption test method, do programmed screening, obtain 3 strain positive hybridoma cell strains and be numbered: 4-1E7,6-2H10,7-1C6.Subgroup identification is carried out in the 3 strain positive cell strains screened, obtains the positive hybridoma cell strain of 3 strain Immunoglobulin IgG1 types.Its titer of ascites can reach 1:51200 and 1:204800, and cell culture supernatant is tired and can be reached 1:256 and 1:512.By the centrifugal 15min of ascites 12 000 × g collected, discard top layer grease, supernatant caprylic acid-ammonium is purified.Still have a small amount of assorted band through sad-ammonium sulfate salting-out process purifying, monoclonal antibody sample is again after G-albumen affinity purification, and almost occur without any assorted band, antibody purity is very high.The monoclonal antibody obtained only with CDV generation specific reaction, and there is no cross reaction with other virus such as parvovirus and canine infectious hepatitis virus-I type (CAV-I) and canine infectious hepatitis virus-II type (CAV-II).
2, against yeasts expresses the preparation of the monoclonal antibody of ermine source parvovirus VP2 gene protein: increase to ermine source parvovirus VP2 gene with polymerase chain reaction method, Successful amplification has arrived parvovirus VP2 gene, by parvovirus VP2 gene clone in Pichia pastoris secretion expression carrier pPICZAA, build recombinant eukaryotic expression plasmid pPICZAA-VP2, after this recombinant plasmid linearization, in transforming Pichia pastoris X-33, methanol induction expresses parvovirus VP2 gene protein, polyacrylamide gel electrophoresis and Western blot qualification expressing protein.Result Successful amplification parvovirus VP2 gene protein, constructs recombinant eukaryotic expression plasmid pPICZAA-VP2, gives expression to about 68 kD albumen in pichia pastoris phaff.After Western blot qualification, protein gene VP2 for the purpose of expressing protein.Expression strain expands expression system and cultivates in nutrient culture media, supernatant 70% ammonium persulfate, 4 DEG C of precipitation concentration, and adopt the VP2 gene protein that His selects the band of the Yeast expression of nickel-affinity chromatography column separating purification acquisition restructuring histidine-tagged, expression is about 3mg/L.With the parvovirus VP2 gene protein immunity Balb/c mouse that the yeast of purifying is recombinant expressed, the recombinant VP 2 gene protein complete Freund's adjuvant after filtering was strengthened primary immune response every two weeks, uses the antigen of incomplete Freund's adjuvant emulsification instead.After generally exempting to exempt from 43,10d detects Balb/c mouse antibodies and tires, when antibody titer reach tire reach more than 1: 5000 ~ 1: 10000 time for merging.Get immune Balb/c mouse spleen and be prepared into splenocyte, with polyglycol PEG1500 for fusion agent and myeloma cell are merged.Choose cell monoclonal, be incubated at 96 porocyte culture plates.Use immunogene wrapper sheet, enzyme-linked immunologic adsorption test method is adopted to the clone selected, do first time screening, obtain 27 strain positive hybridoma cell strains.With the positive hole of limiting dilution assay clone purification, by the cell line of the 27 strain positives, with ermine source parvovirus wrapper sheet again, adopt enzyme-linked immunologic adsorption test method, do programmed screening, obtain 11 strain positive hybridoma cell strains.Screened by enzyme-linked immunologic adsorption test method, obtain the monoclonal antibody hybridoma cell strain of 4 strain energy stably excreting anti-parvovirus VP2 gene protein.In 4 strain monoclonal antibodies, 2 strains belong to Immunoglobulin IgG2 b subclass, and 2 strains belong to Immunoglobulin IgG1 subclass, and its titer of ascites can reach 1:51200 and 1:204800, and cell culture supernatant is tired and can be reached 1:256,1:512.The monoclonal antibody obtained only with parvovirus and parvovirus VP2 gene protein generation specific reaction thereof, and there is no cross reaction with other virus such as CDV and canine infectious hepatitis virus-I type and canine infectious hepatitis virus-II type; Fluorescence immunization coloration Viral diagnosis shows that the specific effect of monoclonal antibody is good further.
3, the preparation of collaurum: adopt trisodium citrate reduction method to prepare collaurum.Measure three of 100mL with volumetric flask and boil off ionized water, pouring specification into is in the flat bottom flask of 500.0mL, is placed on by flask in the heating jacket of magnetic force heating stirrer, put into magnetic stir bar, open and stir knob to suitable speed, open heating knob, be heated to boiling.Add 1.0mL 1% gold chloride (HAuC14) solution, continue heating 2min, then press 100.0mL 0.01 % gold chloride; Add 1.8mL 1% citric acid three sodium solution, continue heating.Lurid aqueous solution of chloraurate after trisodium citrate adds in 2min gradually by xanthochromia ash again blackening finally to redden or orange red.After solution becomes shiny red or is orange red, then continue to add thermal agitation 15min.The collaurum outward appearance of preparation is limpid transparent orange red, and scanning its wavelength X max produced in absorption spectrum corresponding to absorption maximum by spectrophotometer is 522nm, and by electron microscopic observation, even particle size, mean diameter is 18.5nm.
4, ermine source CDV monoclonal antibody (1C6) and parvovirus VP2 gene protein monoclonal antibody (2B3) colloid gold label and purifying.Get a series of small test tube, add 5 mL collaurums respectively, with 0.1 mol/L sal tartari (K
2cO
3) regulate the pH value of collaurum to be adjusted to 6,6.5,7,7.5,8,8.5,9,9.5,10 respectively; Then in the collaurum of often kind of different pH value, add CDV monoclonal antibody (1C6) the 100.0 μ L of 1.0mg/mL respectively, with sealed membrane closed test tube mouth, leave standstill 15min, 4 DEG C of centrifugal l0 min of 2000r/min.Get supernatant ultraviolet one visible spectrophotometer (400 ~ 600 nm) in visible-range to scan, obtain collaurum visible absorption spectrum, measure maximum absorption wavelength, light absorption value.Collaurum pH value corresponding during to occur maximum absorption band is for optimum mark pH value.The optimal pH of CDV monoclonal antibody (1C6) colloid gold label is 8.0, and when in 1mL collaurum, monoclonal antibody addition is 10 μ g, corresponding absorption peak is maximum, and add 20% more on this basis, namely the suitableeest labelled amount is 12 μ g/mL collaurums.Owing to being all mouse resource monoclonal antibody, parvovirus VP2 gene protein monoclonal antibody (2B3) is identical with parvovirus monoclonal antibody (1C6) the suitableeest mark.The collaurum of 20.0mL is added in the small beaker of 50.0mL, after adjusting pH to optimum mark pH value, under the state stirred, slowly add the monoclonal antibody of the good 1mg/mL of a certain amount of dilution, stir 30min, adding 10% bovine serum albumin(BSA) (BSA) to final concentration is 1%, continue stirring 30 min, after 4 DEG C of placement 2h, above-mentioned colloid gold label thing is sub-packed in the centrifuge tube of 7.0mL, with the centrifugal 15min of 2 000r/min, sucking-off supernatant, abandons precipitation; With centrifugal 45 min of 8 700r/min, supernatant discarded night, add mark cleansing solution to original volume; Again with the centrifugal 30min of 10 000r/min, supernatant discarded night, add mark cleansing solution to original volume; With centrifugal 30 min of 10 000r/min, supernatant discarded night, precipitate that to mark cleansing solution with 1.00mL resuspended, put 4 DEG C of refrigerators for subsequent use.
5, nitrocellulose filter and gold conjugation pad bag quilt: 0.01 mol/L pH 7.2 phosphate buffer (PBS) of 6% methyl alcohol is buffered liquid, 0.22um membrane filtration mistake for bag, put 4 DEG C for subsequent use.Confining liquid adopts 2% bovine serum albumin(BSA) (BSA), 1% polysorbas20,0.5% tygon to adjoin pyrrolidone K30 (PVP K30), and 2.5% sucrose, 0.02% nitrine receive (NaN
3), 0.01 mol/L pH7.4 PBS solution, 0.22 μm of miillpore filter filters, put 4 DEG C for subsequent use.Detection film is nitrocellulose filter, selects Millipore HF135, and the flow velocity of film is 135 scholar 34 s/cm, and aperture is approximately 0.45 μm.After first anti-for rabbit ermine source CDV and parvovirus antibody, rabbit anti-mouse antibody being buffered liquid dilution with described bag, be sprayed on described nitrocellulose filter Millipore HF135 respectively, as detection line 10(hundstaupe hot line and parvovirus line) and nature controlling line 11, being sprayed on the rabbit anti-ermine source CDV antibody concentration detecting hundstaupe hot line on film nitrocellulose filter detection line 10 is 1.5mg/mL, and quantity for spray is 2-3 μ L/cm; Being sprayed on the rabbit anti-ermine source parvovirus antibody concentration detecting parvovirus line on film nitrocellulose filter detection line 10 is 1.0mg/mL, and quantity for spray is 3-4 μ L/cm; Being sprayed on and detecting rabbit anti-mouse antibody concentration on film nitrocellulose filter nature controlling line 11 is 0.8mg/mL, and quantity for spray is 1-3 μ L/cm, in 37 DEG C of incubator dry for standby.The material of long and short gold conjugation pad is glass fibre membrane, after the anti-ermine source CDV monoclonal antibody of colloid gold label and the dilution of parvovirus VP2 gene protein monoclonal antibody, be sprayed on long gold conjugation pad 8 and short gold conjugation pad 9 respectively, 37 DEG C of dry for standby.5 times of dilutions are done in colloid gold label anti-ermine source CDV monoclonal antibody (1.5mg/mL) and short gold conjugation pad anti-ermine source parvovirus VP2 gene protein monoclonal antibody (1.5mg/mL) that are sprayed on long gold conjugation pad, and quantity for spray is respectively 50-60 μ L/cm.
6, the assembling of test paper: by the sample pad 6 handled well, be coated with the long gold conjugation pad 8 of anti-ermine source CDV and parvovirus VP2 gene protein monoclonal antibody-colloid gold label thing, short gold conjugation pad 9, be coated with rabbit anti-ermine source CDV and parvovirus antibody as detection line 10 be coated with the nitrocellulose filter 5 of rabbit anti-mouse antibody as nature controlling line 11, absorption pad 7 order sticks on rigid polyvinyl chloride liner plate 4 successively, composition test strips, have in the test strips cartridge 3 of well 1 and colour developing district opening 2 above this test strips is arranged on, Vacuum Package, normal temperature is preserved, the term of validity 6 months.
The present invention can detect the application of fox, racoon dog, the canine distemper of ermine and the immunochromatography colloid gold test paper of parvovirus simultaneously:
1, the pre-service of fox, racoon dog, ermine sample: the fox of the doubtful canine distemper collected, racoon dog, ermine sample such as the tears of sick fox, racoon dog, ermine, nose are wiped and the fox of parvovirus infections, racoon dog, ermine ight soil or the fox died of illness, racoon dog, ermine internal organs sample, respectively have negative sample to compare.The pathological material of disease of doubtful CDV fox, racoon dog, ermine as tears, nose wipe, every part, internal organs sample of dying of illness gets about 0.5g(mL) left and right, add physiological saline and be about 0.5mL, make suspension.Every part, the ight soil of the fox of doubtful parvovirus infections, racoon dog, ermine gets about about 0.5g, adds physiological saline and is about 0.5mL, fully leaves standstill 5min (or centrifugal) after shake.
2, detect: get supernatant 100 μ L respectively and drop on test paper of the present invention, observations after 10min.Get 100 μ L 0.01M PBS and known TCID respectively simultaneously
50be 9.4 × 10
5cDV, parvovirus cell culture fluid (1:512 dilution) in the well of another two test paper, do negative blank and positive criteria product control test.
3, result judges: when nature controlling line 11 presents red line, the fox of doubtful canine distemper, racoon dog, ermine sample is added in well 1, at colour developing opening 2 place of district, there is red line in detection line 10, for canine distemper is positive, namely contain CDV in sample, do not occur red line, for canine distemper is negative, namely do not contain CDV in sample; In well 1, add the sample of the fox of doubtful parvovirus infections, racoon dog, ermine, there is red line in detection line 10, for parvovirus is positive, namely parvovirus is contained in sample, there is not red line, for parvovirus is negative, namely do not contain parvovirus in sample.If red line does not appear in nature controlling line 11, then test paper is invalid.
Effect citing of the present invention:
1, specific test: the CDV know oneself and parvovirus cell culture fluid carry out cross matching, establish PBS negative control simultaneously.With the TCID that 0.01M PBS knows oneself
50be 9.4 × 10
5cDV and parvovirus cell culture fluid make doubling dilution to 1:512, get 100 μ L respectively and drop in well, use detection paper of the present invention, carry out sensitivity tests.When containing CDV and parvovirus in sample simultaneously, in colour developing district opening 2, detection line 10(two red detection lines) be all positive (having 2 red stripes to occur), and when only containing CDV or parvovirus in sample, on a detection line of correspondence, then only present positive reaction (being equipped with 1 red line in corresponding positions to occur), PBS negative control (detection line occurs without any band) and other virus such as canine infectious hepatitis virus-I type and canine infectious hepatitis virus-II type are all negative reaction (all without band occur), come to the same thing after repeatedly repeating 5 times, illustrate that the method has the specificity of height.
2, sensitivity tests: with PBS to known CDV or parvovirus (cell median infective dose TCID
50be 9.4 × 10
5) make doubling dilution to 1:512, get 100 μ L respectively and drop in well, test paper is still positive (detection line has 2 red stripes to occur), proves that the susceptibility of test paper of the present invention is strong.
3, stability test: after 5 batches of test paper of the present invention (batch number 120516,120518,120519,120528,120530) are placed 7d in 37 DEG C of constant incubators, detect 10 parts of CDVs and parvovirus positive and 10 parts of CDVs and parvovirus negative sample with same batch of test paper of preservation 4 DEG C simultaneously, result show: 37 DEG C effect 7d to test paper of the present invention without destruction, prove that test paper is at high temperature stablized.
4, storage life test: by the test paper of the present invention with a collection of preparation, respectively 4 DEG C and room temperature preservation 1 to 6 month, took out in 0 month, 3 months, 6 months, the CDV and the parvovirus positive that detect 1 part of doubling dilution carry out storage life sensitivity tests, detect 10 parts of known CDVs and parvovirus positive and CDV and parvovirus negative sample and carry out storage life specific test, observation period susceptibility, specificity and stability, to determine the storage life of test paper.Result shows: test paper of the present invention does not all change through 6 months phases of room temperature preservation specificity, susceptibility.
5, with enzyme linked immunosorbent assay, hemagglutination test Measures compare: the canine distemper of fox, racoon dog, ermine is made a definite diagnosis conventional enzyme linked immunosorbent assay and detected clinically, the parvovirus of fox, racoon dog, ermine makes a definite diagnosis the detection of conventional hemagglutination test method.In the tears of 120 parts of tested canine distemper suspected case foxes, racoon dog, ermine (nose is wiped, internal organs of dying of illness) sample, detecting the positive by enzyme linked immunosorbent assay is 47 parts, feminine gender is 73 parts, it is 49 parts by the detection paper positive of the present invention, feminine gender is 71 parts, result shows: canine distemper test paper method Positive rate 40.83% of the present invention comparatively enzyme linked immunosorbent assay detection Positive rate 39.16% height (test findings detects with polymerase chain reaction method simultaneously, conforms to); In ight soil (internal organs of the dying of illness) sample of 120 parts of tested parvovirus suspected case foxes, racoon dog, ermine, detecting the positive by blood coagulation tests method is 50 parts, feminine gender is 70 parts, it is 53 parts by the detection paper positive of the present invention, feminine gender is 67 parts, result shows: parvovirus test paper method Positive rate 79.10% of the present invention comparatively hemagglutination test experimental technique detection Positive rate 71.42% height (test findings detects with polymerase chain reaction method simultaneously, conforms to).
Claims (2)
1. can detect fox, racoon dog, the canine distemper of ermine and an immune colloid gold test paper for parvovirus simultaneously, the test strips cartridge of well and colour developing district opening is set above being included in, and be arranged on the test strips in this test strips cartridge, described test strips includes rigid polyvinyl chloride liner plate, nitrocellulose filter, sample pad and absorption pad, it is characterized in that: described rigid polyvinyl chloride liner plate is arranged on the box bottom surface of described test strips cartridge, described nitrocellulose filter is placed in above described rigid polyvinyl chloride liner plate, the long gold conjugation pad be placed in above described nitrocellulose filter and the short gold conjugation pad be placed in above described long gold conjugation pad is provided with in one end of described test strips cartridge, described sample pad is placed in above described short gold conjugation pad, corresponding with described well above this sample pad, the other end that described absorption pad is placed in described test strips cartridge is positioned at above described nitrocellulose filter, described long gold conjugation pad and short gold conjugation pad are coated with respectively the VP2 gene protein monoclonal antibody-colloid gold label thing of anti-ermine source CDV and parvovirus, described anti-ermine source CDV and the VP2 gene protein monoclonal antibody of parvovirus are the VP2 gene protein immunity Balb/c mouse of the parvovirus using the band of the ermine source CDV of purifying and the Yeast expression of restructuring histidine-tagged, through Fusion of Cells, the VP2 gene protein monoclonal antibody that screening obtains anti-ermine source CDV and parvovirus is that hybridoma cell line is secreted, described nitrocellulose filter is coated with respectively two detection lines of rabbit anti-ermine source CDV and parvovirus antibody formation and a nature controlling line of rabbit anti-mouse antibody formation, these two detection lines are corresponding with described colour developing district opening with nature controlling line.
2. the preparation method that simultaneously can detect fox, racoon dog, the canine distemper of ermine and the immune colloid gold test paper of parvovirus according to claim 1, is characterized in that: it comprises the following steps:
(1) the VP2 gene protein of the histidine-tagged parvovirus of the band of the ermine source CDV of purifying and the Yeast expression of restructuring is prepared;
(2) the VP2 gene protein immunity Balb/c mouse of the parvovirus using the band of the ermine source CDV of purifying and the Yeast expression of restructuring histidine-tagged respectively, through Fusion of Cells, screening obtains the hybridoma cell line of the VP2 gene protein monoclonal antibody of secreting anti-ermine source CDV and parvovirus;
(3) the VP2 gene protein monoclonal antibody of anti-ermine source CDV and parvovirus is prepared;
(4) react with trisodium citrate and gold chloride and prepare collaurum;
(5) the VP2 gene protein monoclonal antibody of anti-ermine source CDV obtained for step (3) and parvovirus is added respectively in the collaurum that step (4) prepares, obtain the VP2 gene protein monoclonal antibody-colloid gold label thing of anti-ermine source CDV and parvovirus respectively;
(6) the VP2 gene protein monoclonal antibody-colloid gold label thing of anti-ermine source CDV and parvovirus is sprayed on long gold conjugation pad and short gold conjugation pad respectively;
(7) rabbit anti-ermine source CDV and parvovirus antibody is prepared, preparation rabbit anti-mouse antibody;
(8) antibody of anti-for rabbit ermine source CDV and parvovirus is sprayed on formation two detection lines on nitrocellulose filter, and rabbit anti-mouse antibody is sprayed on a formation nature controlling line on nitrocellulose filter;
(9) rigid polyvinyl chloride liner plate, nitrocellulose filter, long gold conjugation pad, short gold conjugation pad, sample pad, absorption pad are bonded together composition test strips in order, and load fixing on request for this test strips in test strips cartridge, obtain the described immune colloid gold test paper that simultaneously can detect fox, racoon dog, ermine canine distemper and parvovirus.
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| CN109652597A (en) * | 2019-01-26 | 2019-04-19 | 沈阳农业大学 | It is a kind of for detect dog, cat, ermine parvovirus general RPA primer, RPA probe, kit and nucleic acid detection method |
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Address after: 066600 College of animal science and technology, Hebei Normal University of Science and Technology, Changli, Hebei, Qinhuangdao Patentee after: Hebei Science & Technology Normal College Patentee after: HEBEI XINHUA KEJI VETERINARY MEDICINE GROUP CO., LTD. Address before: 066600 College of animal science and technology, Hebei Normal University of Science and Technology, Changli, Hebei, Qinhuangdao Patentee before: Hebei Science & Technology Normal College Patentee before: Hebei Xinhua Keji Veterinary Medicine Co., Ltd. |