CN104459119B - A kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof - Google Patents
A kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof Download PDFInfo
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- CN104459119B CN104459119B CN201410642350.8A CN201410642350A CN104459119B CN 104459119 B CN104459119 B CN 104459119B CN 201410642350 A CN201410642350 A CN 201410642350A CN 104459119 B CN104459119 B CN 104459119B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
Abstract
The invention discloses a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof, comprise sample pad, pad, nitrocellulose membrane, adsorptive pads and base plate, feature is stained with sample pad successively in order on base plate, pad, nitrocellulose membrane, adsorptive pads, this pad is coated with described anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing, this nitrocellulose membrane is coated with respectively the detection line of soft-shelled turtle systemic septicaemia ball viral antigen formation and the nature controlling line of the anti-chicken yolk antibody formation of rabbit, advantage is highly sensitive, high specificity, easy and simple to handle, detect fast, accuracy is high.
Description
Technical field
The present invention relates to immunochemistry detection technique, especially relate to a kind of Test paper in order to detect soft-shelled turtle systemic septicaemia virus in the various tissue of animal, blood and ight soil fast based on Yolk antibody, colloidal gold immunochromatographimethod technology and preparation method thereof.
Background technology
Soft-shelled turtle is one of the most well-known nourishing and body-strengthening treasure, and whole body all can be used as medicine, and is the traditional Chinese crude drug of China, has higher economic worth.Along with living standards of the people improve and going deep into edible, the medical value understanding of soft-shelled turtle day by day, the demand of soft-shelled turtle constantly increases, and propagates the application of soft-shelled turtle industry artificially and gives birth to.But along with the expansion of cultivation scale, disease is broken out again and again, grows in intensity, wherein main based on soft-shelled turtle systemic septicaemia.
Soft-shelled turtle systemic septicaemia is a kind of eruptive epidemic disease betiding soft-shelled turtle ecological cultivation farm, and 6-September is its onset peak period, and it breaks with tremendous force, and in 7-10 days, morbidity pond cumulative mortality can reach 90-100%.Sick soft-shelled turtle is mainly the young soft-shelled turtle of body weight 150-200g, becomes soft-shelled turtle also to have ill infection.The primary cause of disease of this disease is soft-shelled turtle systemic septicaemia spherical viruses (Soft-shelledturtlesystemicsepticemiasphericalvirus, STSSSV) (China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, preserving number is CGMCCNo.5378), ill soft-shelled turtle cardinal symptom is hemorrhagic sepsis, there is a large amount of spherical viruses particle in the tissue such as the heart, liver, stomach.Through investigation, soft-shelled turtle systemic septicaemia is once outburst, and routine medication is invalid, carrys out tremendous economic loss to cultivation industrial zone.Therefore, detect at seed purchase, introduction and their early stage, determine the state of an illness, to take measures in time to control to infect and be worse off, to minimizing, plant loses significant.
At present, the detection method of pathogen mainly contains and is separated cultivation, electron microscopic observation, PCR, ELISA, stratographic analysis etc., and these methods take time and effort more, need special place and instrument and equipment, need professional to operate.Researchist successfully establishes the technology that ELISA detects soft-shelled turtle systemic septicaemia virus (STSSSV), but whole testing process at least needs a few hours, and requires higher to operator, needs the professional equipment that microplate reader is such.
Colloidal gold immunochromatographimethod (gold-immunochromatographyassayGICA) is the new immunoassay formats starting to grow up the eighties in 20th century, it is application colloidal gold-labeled method, using collaurum as tracer, based on a kind of Novel immune labelling technique of antigen-antibody reaction, it has easy, fast, and high specificity, highly sensitive, the advantage that expense is low.According to colloidal gold immunochromatographimethod technology, at home and abroad no matter people cures application aspect, or animal doctor applies conveniently, all have developed the test strips that multiple colloidal gold immunochromatographimethod detects various cause of disease and poisonous and harmful substance fast.But, any correlative study about soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof report is not also disclosed both at home and abroad at present.
Summary of the invention
Soft-shelled turtle systemic septicaemia Viral diagnosis test strips that technical matters to be solved by this invention is to provide a kind of high specificity based on colloidal gold immunochromatographimethod technology, highly sensitive, detection speed is fast, cost is low and easy and simple to handle and preparation method thereof, meets the demand of field quick detection.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips, and described test strips is by bar shaped bottom plate and the sample pad overlapped successively on bar shaped bottom plate, pad, nitrocellulose filter and adsorptive pads; Described pad is coated with anti-soft-shelled turtle systemic septicaemia virus Yolk antibody-colloid gold label thing, described nitrocellulose filter is respectively arranged with by the detection line of soft-shelled turtle systemic septicaemia viral antigen bag quilt and the nature controlling line being wrapped quilt by the anti-chicken yolk antibody of rabbit (IgY).
The preparation method of described anti-soft-shelled turtle systemic septicaemia virus Yolk antibody-colloid gold label thing is as follows: the colloidal gold solution being 15-30nm by anti-soft-shelled turtle systemic septicaemia virus Yolk antibody and colloid gold particle diameter mixes by 6-8 μ g:1mL, under the condition of pH5.4, make it combine by stirring vibration 30-60min, add PBST damping fluid containing the bovine serum albumin(BSA) of 10wt% as stabilizing agent, adopt low-speed centrifugal 2000-3500rpm, 10-20min removes fully unstable colloid gold particle and condensation product thereof, high speed centrifugation 12000-15000rpm again, 1-1.5h removes unconjugated anti-soft-shelled turtle systemic septicaemia virus Yolk antibody, get kermesinus precipitation bottom centrifuge tube and namely obtain anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing.
A preparation method for soft-shelled turtle systemic septicaemia Viral diagnosis test strips, comprises the following steps:
(1) preparation of sample pad
Be soaked in by all-glass paper in the PBS damping fluid containing the 0.01MpH7.4 of 1wt% bovine serum albumin(BSA), 2.5wt% sucrose, 1wt% trehalose, 1wt% Tween-20 and take out after 30min, namely obtain sample pad, Vacuum Package in 37 DEG C of dry 2-3h, 4 DEG C save backup;
(2) preparation of the pad of specified packet quilt
All-glass paper is soaked in containing 1wt% bovine serum albumin(BSA), 2.5wt% sucrose, 1wt% trehalose, in the PBS damping fluid of the 0.01MpH7.4 of 1wt% Tween-20 after 30min, after 37 DEG C of dry 1-2h, the anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing of even application 10-20 μ L on the all-glass paper of every square centimeter, vacuum freeze drying 1-2h, namely obtain pad, Vacuum Package, 4 DEG C save backup;
(3) preparation of the nitrocellulose filter of specified packet quilt
Soft-shelled turtle systemic septicaemia spherical viruses antigen point film instrument is coated on nitrocellulose membrane and is formed detection line; Be coated on nitrocellulose membrane by anti-for rabbit chicken yolk antibody point film instrument and form nature controlling line, namely obtain the nitrocellulose membrane of specified packet quilt, vacuum freeze drying 1-1.5h, Vacuum Package, 4 DEG C save backup; Wherein detection line and nature controlling line are parallel to each other, and described detection line is near pad end, and described nature controlling line is near adsorptive pads end;
(4) preparation of test strips
The nitrocellulose membrane of the specified packet quilt that the pad of the specified packet quilt that sample pad step (1) obtained, step (2) obtain, step (3) obtain and adsorptive pads overlap according to the order of sequence and are pasted on base plate, be cut into the slice that 4-6mm is wide, obtain soft-shelled turtle systemic septicaemia Viral diagnosis test strips, Vacuum Package, 4 DEG C of preservations.
The preparation method of described soft-shelled turtle systemic septicaemia spherical viruses antigen is as follows: get and attack the little soft-shelled turtle that poison infects death, solution takes its internal organ on ice, weigh, by 1:5-1:20 add that ice bath in advance crosses containing homogenate in the TNE buffer solution of 1mM phenylmethylsulfonyl fluoride (PMSF), by homogenate in 4 DEG C, the centrifugal 20min of 8000g, precipitation with containing 1mMPMSF the homogenate of TNE damping fluid Eddy diffusion after again in 4 DEG C, the centrifugal 20min of 8000g, merge twice centrifugal gained supernatant, by supernatant in 4 DEG C, after the centrifugal 10min of 6000g, get supernatant in 4 DEG C, the centrifugal 1.5h of 38000g, wash away precipitation upper strata with the TM damping fluid of precooling to loosen part, take off layer solid white precipitate part with after resuspended containing the TM damping fluid of precooling of 1mMPMSF in 4 DEG C, after the centrifugal 5min of 3500g, get supernatant again in 4 DEG C, the centrifugal 1.5h of 38000g, wash away upper strata with TM damping fluid to loosen part, in 4 DEG C after the solid white precipitate part of lower floor is again resuspended with the TM damping fluid of the precooling containing 1mMPMSF, after the centrifugal 5min of 3500g, get supernatant again in 4 DEG C, the centrifugal 1.5h of 38000g, wash away upper strata with TM damping fluid to loosen part, the solid white precipitate of the lower floor finally obtained is soft-shelled turtle systemic septicaemia spherical viruses antigen.
The preparation method of described anti-soft-shelled turtle systemic septicaemia virus Yolk antibody-colloid gold label thing is as follows:
(1) preparation of anti-soft-shelled turtle systemic septicaemia virus Yolk antibody
Get healthy first hen of laying eggs as immunization, by soft-shelled turtle systemic septicaemia viral antigen and the mixing of Freund's complete adjuvant equal-volume, the fully emulsified rear dipteron to hen, both legs, fundamental immunity is carried out in back and the intramuscular injection of chest muscle position, every hen immunizing dose is 500-800 μ g/mL, after 15-20 days, soft-shelled turtle systemic septicaemia viral antigen and incomplete Freund's adjuvant are carried out equal-volume mixing, fully emulsifiedly rear booster immunization is carried out to hen intramuscular injection, every hen immunizing dose is 400-600 μ g/mL, booster immunization repeats three times, each interval time is 7-10 days, then collect egg mensuration to tire, egg is collected when Yolk antibody tires >1:20000, be separated yolk, Yolk antibody is purified by saturated ammonium sulphate method,
(2) colloid gold label anti-soft-shelled turtle systemic septicaemia virus Yolk antibody
The colloidal gold solution being 15-30nm by anti-soft-shelled turtle systemic septicaemia virus Yolk antibody and colloid gold particle diameter mixes by 6-8 μ g:1mL, under the condition of pH5.4, make it combine by stirring vibration 30-60min, add PBST damping fluid containing the bovine serum albumin(BSA) of 10wt% as stabilizing agent, adopt low-speed centrifugal 2000-3500rpm, 10-20min removes fully unstable colloid gold particle and condensation product thereof, high speed centrifugation 12000-15000rpm again, 1-1.5h removes unconjugated anti-soft-shelled turtle systemic septicaemia virus Yolk antibody, get kermesinus precipitation bottom centrifuge tube and namely obtain anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing, by described anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing with containing 1wt% bovine serum albumin(BSA), 2.5wt% sucrose, 1wt% trehalose, the resuspended 1/10-1/20 to described colloidal gold solution volume of 0.01MPBS damping fluid of the pH7.4 of 0.02wt% sodium azide is as its working concentration.
The concrete grammar of Yolk antibody is purified as follows: get step (1) and be separated the yolk obtained by saturated ammonium sulphate method, 1:9 adds the acetic acid-sodium acetate buffer solution of the 0.05M of pH5.0 by volume, stir rear 4 DEG C of hold over night, the centrifugal 20min of 8000g, getting supernatant, to add saturated ammonium sulfate to saturation degree be 40%, 4 DEG C are stirred 6h, the centrifugal 20min of 10000g, the DDW getting precipitation 10-20 times of yolk quality is resuspended, adding saturated sodium sulphate to saturation degree is 40%, 4 DEG C of stirrings are spent the night, the centrifugal 20min of 10000g, precipitate resuspended with the PBS damping fluid of the 0.01M of a small amount of pH7.4 after, dialysed overnight in PBS damping fluid, namely anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody is obtained.
In step (2), colloidal gold solution preparation method is as follows: by the chlorauric acid solution of 100mL0.01wt%, after heating is boiled, add 1-2mL1wt% trisodium citrate while stirring to shake up rapidly, continue heating, solution becomes claret by faint yellow blackening, continues heating 5-10min, supply dehydration to 100mL after cooling to colour stable, sterile sealing, 4 DEG C keep in Dark Place.
Described detection line is apart from described pad 6-8mm; Described nature controlling line is apart from described adsorptive pads 6-8mm, and the width of described detection line and described nature controlling line is respectively 1-1.5mm, and two lines are apart 5mm.
Described TNE buffer method is as follows: Tris6.0578g, NaCl12g, disodium ethylene diamine tetraacetate 1.8612g, supplies DDW to 1L, regulates pH to 8.5, autoclaving;
Described TM buffer method is as follows: Tris6.0578g, MgCl
26H
2o2.0338g, supplies DDW to 1L, regulates pH to 7.4, autoclaving;
Described PBST buffer method is as follows: NaCl8g, KCl0.2g, Na
2hPO
412H
2o2.9g, KH
2pO
40.2g, 500 μ L Tween-20s, supply DDW to 1L, regulate pH to 7.4, autoclaving;
Described PBS buffer method is as follows: NaCl8g, KCl0.2g, Na
2hPO
412H
2o2.9g, KH
2pO
40.2g, supplies DDW to 1L, regulates pH to 7.4, autoclaving;
Described acetic acid-sodium acetate buffer solution formula is as follows: sodium acetate 2.604g, glacial acetic acid 1.095g, supplies DDW to 1L, adjusts pH to 5.0, autoclaving.
The package amount of described soft-shelled turtle systemic septicaemia spherical viruses antigen is 2-4 μ L/cm; The package amount of the anti-chicken yolk antibody of described rabbit is 1-2 μ L/cm, is that the phenylmethylsulfonyl fluoride of 1mM and the trehalose of 5wt% are as protease inhibitors and antigen protective agent containing concentration in described soft-shelled turtle systemic septicaemia spherical viruses antigen.
The present invention selects soft-shelled turtle systemic septicaemia spherical viruses as detection line, whether anti-soft-shelled turtle systemic septicaemia spherical viruses special yolk antibody, as the antibody of colloid gold label, utilizes competition law to detect in testing sample containing soft-shelled turtle systemic septicaemia spherical viruses.By the soft-shelled turtle systemic septicaemia spherical viruses in measuring samples and the common competition binding of the soft-shelled turtle systemic septicaemia spherical viruses antigen anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing be coated on nitrocellulose membrane.If the amount of soft-shelled turtle systemic septicaemia spherical viruses is higher than the detectability of test strips in measuring samples, soft-shelled turtle systemic septicaemia spherical viruses in sample is all combined anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing, thus does not occur red stripes and aobvious positive not with being fixed on the virus on nitrocellulose membrane and being combined; If do not measure the detectability lower than test strips containing soft-shelled turtle systemic septicaemia spherical viruses or its in measuring samples, anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing all can not be combined by soft-shelled turtle systemic septicaemia spherical viruses in sample, and the virus that so golden labeling antibody can be fixed on nitrocellulose membrane in chromatography process combines and occurs red stripes and show negative.Therefore, if the detection line of testing sample test strips and nature controlling line occur red stripes simultaneously, then negative sample is judged as; If testing sample ELISA test strip line does not occur red stripes, nature controlling line occurs that red stripes is then judged as positive simultaneously; If nature controlling line does not have red stripes occur, then this test strips is invalid.
Compared with prior art, the invention has the advantages that
1, detect fast: whole testing process only needs 5-10 minute, can meet the needs of Site Detection;
2, high, the high specificity of Detection accuracy: this reaction and other viruses do not have cross reaction, and detection accuracy can reach more than 95%, comes to the same thing with the ELISA of complicated operation;
3, easy to carry, easy and simple to handle: the present invention changes must by professional people to the detection of soft-shelled turtle systemic septicaemia spherical viruses
4, test strip preparation technology of the present invention is simple, with low cost, without any need for specific apparatus, equipment;
5, test strip of the present invention stores conveniently, not high to temperature requirement, effectively can preserve half a year more than at 4 DEG C;
6, test strip of the present invention not only may be used for each tissue detecting animal, also may be used for detecting blood and ight soil.
Soft-shelled turtle systemic septicaemia spherical viruses (Soft-shelledturtlesystemicsepticemiasphericalvirus, STSSSV), strain is P1015 strain, preserving number is CGMCCNo.5378, China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited on October 21st, 2011, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Accompanying drawing explanation
Fig. 1 is the structural representation of test strips of the present invention, and in figure, 1 is sample pad, and 2 is pad, and 3 is nitrocellulose filter, and 4 is absorption pad, and 5 is detection line, and 6 is nature controlling line, and 7 is bar shaped bottom plate;
Fig. 2 is preparation technology's process flow diagram of test strips of the present invention.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment 1
A kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips, as shown in Figure 1, this test strips is by bar shaped bottom plate 7 and the sample pad 1 overlapped successively on bar shaped bottom plate 7, pad 2, nitrocellulose filter 3 and adsorptive pads 4; Pad 2 is coated with anti-soft-shelled turtle systemic septicaemia virus Yolk antibody-colloid gold label thing, nitrocellulose filter 3 is respectively arranged with by the detection line 5 of soft-shelled turtle systemic septicaemia viral antigen bag quilt and the nature controlling line 6 being wrapped quilt by the anti-chicken yolk antibody of rabbit (IgY).
The preparation method of above-mentioned anti-soft-shelled turtle systemic septicaemia virus Yolk antibody-colloid gold label thing is as follows: mixed by 6-8 μ g:1mL with the colloidal gold solution of colloid gold particle diameter 15-30nm by anti-soft-shelled turtle systemic septicaemia virus Yolk antibody, under the condition of pH5.4, make it combine by stirring vibration 30-60min, add PBST damping fluid containing the bovine serum albumin(BSA) of 10wt% as stabilizing agent, adopt low-speed centrifugal 2000-3500rpm, 10-20min removes fully unstable colloid gold particle and condensation product thereof, high speed centrifugation 12000-15000rpm again, 1-1.5h removes unconjugated anti-soft-shelled turtle systemic septicaemia virus Yolk antibody, get kermesinus precipitation bottom centrifuge tube and namely obtain anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing.
Specific embodiment 2
The preparation method of quick detection soft-shelled turtle systemic septicaemia spherical viruses colloidal gold strip, as shown in Figure 2, its concrete operation step is as follows:
(1) purifying of soft-shelled turtle systemic septicaemia spherical viruses
Get and attack the little soft-shelled turtle that poison infects death, solution takes its internal organ on ice, after weighing by volume (1:5)-(1:20) add that ice bath in advance crosses containing homogenate in the TNE buffer solution of 1mM phenylmethylsulfonyl fluoride (PMSF), by homogenate in 4 DEG C, the centrifugal 20min of 8000g, precipitation with containing 1mMPMSF the homogenate of TNE damping fluid Eddy diffusion after again in 4 DEG C, the centrifugal 20min of 8000g, merge twice centrifugal gained supernatant, by supernatant in 4 DEG C, after the centrifugal 10min of 6000g, get supernatant in 4 DEG C, the centrifugal 1.5h of 38000g, wash away precipitation upper strata with precooling TM damping fluid to loosen part, take off layer solid white precipitate part with after resuspended containing the TM damping fluid of precooling of 1mMPMSF in 4 DEG C, after the centrifugal 5min of 3500g, get supernatant again in 4 DEG C, the centrifugal 1.5h of 38000g, wash away upper strata with TM damping fluid to loosen part, in 4 DEG C after the solid white precipitate of lower floor is again resuspended with the TM damping fluid of the precooling containing 1mMPMSF, after the centrifugal 5min of 3500g, get supernatant again in 4 DEG C, the centrifugal 1.5h of 38000g, wash away upper strata with TM damping fluid to loosen part, the solid white precipitate of the lower floor finally obtained is soft-shelled turtle systemic septicaemia spherical viruses antigen.This soft-shelled turtle systemic septicaemia spherical viruses (Soft-shelledturtlesystemicsepticemiasphericalvirus, STSSSV), strain is P1015 strain, preserving number is CGMCCNo.5378, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 21st, 2011;
(2) preparation of anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody
Get healthy first hen of laying eggs as immunization, by soft-shelled turtle systemic septicaemia spherical viruses antigen and the mixing of Freund's complete adjuvant equal-volume, the fully emulsified rear dipteron to hen, both legs, fundamental immunity is carried out in back and the intramuscular injection of chest muscle position, every hen immunizing dose is 500-800 μ g/mL, after 15-20 days, soft-shelled turtle systemic septicaemia spherical viruses antigen and incomplete Freund's adjuvant are carried out equal-volume mixing, fully emulsifiedly rear booster immunization is carried out to hen intramuscular injection, every hen immunizing dose is 400-600 μ g/mL, booster immunization repeats three times, each interval time is 7-10 days, collect egg mensuration after carrying out three immunity to tire, egg is collected when Yolk antibody tires >1:20000, be separated yolk, Yolk antibody is purified by saturated ammonium sulphate method.The concrete grammar of the purifying of Yolk antibody is as follows: take appropriate yolk, acetic acid-sodium acetate buffer solution (0.05M is added by 1:9, pH5.0), stir rear 4 DEG C of hold over night, the centrifugal 20min of 8000g, getting supernatant, to add saturated ammonium sulfate to saturation degree be 40%, 4 DEG C are stirred 6h, the centrifugal 20min of 10000g, precipitate with initial yolk quality 10-20 distilled water doubly resuspended, adding saturated sodium sulphate to saturation degree is 40%, 4 DEG C of stirrings are spent the night, the centrifugal 20min of 10000g, get a small amount of PBS solution (0.01M of precipitation, pH7.4) resuspended, dialysed overnight in PBS solution, namely anti-soft-shelled turtle systematicness spherical viruses Yolk antibody is obtained,
(3) method of colloid gold label anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody
By trisodium citrate reduction gold chloride legal system for colloidal gold solution.With the gold chloride of the deionized water preparation 100mL0.01% of brand-new, after heating is boiled, add 2mL1% trisodium citrate while stirring to shake up rapidly, continue heating, solution becomes claret by faint yellow blackening, continues heating 5-10min, supply dehydration to 100mL after cooling to colour stable, sterile sealing, 4 DEG C keep in Dark Place;
The colloidal gold solution being 15-30nm by anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody and colloid gold particle diameter mixes by 6-8 μ g:1mL, under the condition of pH5.4, make it combine by stirring vibration 30-60min, add PBST damping fluid containing the bovine serum albumin of 10wt% as stabilizing agent, adopt low-speed centrifugal (2000-3500rpm, 10-20min) remove fully unstable colloid gold particle and condensation product thereof, high speed centrifugation (12000-15000rpm again, 1-1.5h) remove unconjugated Yolk antibody, get kermesinus precipitation bottom centrifuge tube and namely obtain anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing, by described anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing with containing 1wt% bovine serum albumin(BSA), 2.5wt% sucrose, 1wt% trehalose, the resuspended 1/10-1/20 to colloidal gold solution volume of 0.01MPBS damping fluid of the pH7.4 of 0.02wt% sodium azide is as its working concentration,
(4) the bag quilt of pad
All-glass paper is soaked in containing 1wt% bovine serum albumin(BSA), 2.5wt% sucrose, 1wt% trehalose, in the PBS damping fluid of the 0.01MpH7.4 of 1wt% Tween-20 after 30min, after 37 DEG C of dry 1-2h, even application 10-20 μ L anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing on the all-glass paper of every square centimeter, vacuum freeze drying 1-2h, namely obtain pad, Vacuum Package, 4 DEG C save backup;
(5) process of sample pad
All-glass paper is soaked in the PBS damping fluid containing the 0.01MpH7.4 of 1wt% bovine serum albumin(BSA), 2.5wt% sucrose, 1wt% trehalose, 1wt% Tween-20 and takes out after 30min, namely 37 DEG C of dry 2-3h obtain sample pad, Vacuum Package, 4 DEG C save backup;
(6) the bag quilt of nitrocellulose membrane
Be coated on nitrocellulose membrane by soft-shelled turtle systemic septicaemia spherical viruses antigen point film instrument and form detection line, package amount is 2-4 μ L/cm; Be coated on nitrocellulose membrane by anti-for rabbit chicken yolk antibody point film instrument and form nature controlling line, package amount is 1-2 μ L/cm, namely obtains the nitrocellulose filter of specified packet quilt, vacuum freeze drying 1-1.5h, Vacuum Package, and 4 DEG C save backup; Wherein detection line and nature controlling line are parallel to each other, and described detection line is near pad end, and described nature controlling line is near adsorptive pads end; Containing concentration in soft-shelled turtle systemic septicaemia spherical viruses antigen is the phenylmethylsulfonyl fluoride (protease inhibitors) of 1mM and the trehalose (antigen protective agent) of 5wt%;
(7) test strips assembling
Sample pad (1), pad (2), nitrocellulose membrane (3), adsorptive pads (4) are sticked on bar shaped bottom plate (7) by the order shown in Fig. 1 successively, bar shaped bottom plate is as prop carrier, by lower and on the absorption of sample district that is made up of all-glass paper, then be the glass layer having adsorbed the Yolk antibody that collaurum marks, next is nitrocellulose membrane, be the backing film that a macromolecular material is made between nitrocellulose membrane and bar shaped bottom plate, stop organic solvent in bonding agent to the destruction of institute's coated antibody on nitrocellulose membrane.Most last layer is water accepting layer, and be made up of filter paper, outside adhesive tape seals, and becomes portion of the handle.The overlap of about 1.5mm is all had to intersect between each layer.Test strips is cut into the little bar that 4-6mm is wide, Vacuum Package, 4 DEG C of preservations.
The TNE buffer method wherein adopted in step (1) is: Tris6.0578g, NaCl12g, disodium ethylene diamine tetraacetate 1.8612g supply DDW to 1L, regulates pH to 8.5, autoclaving; TM buffer method is: Tris6.0578g, MgCl
26H
2o2.0338g supplies DDW to 1L, regulates pH to 7.4, autoclaving.
The PBS buffer method adopted in step (2) (3) (4), (5) is: NaCl8g, KCl0.2g, Na
2hPO
412H
2o2.9g, KH
2pO
40.2g supplies DDW to 1L, regulates pH to 7.4, autoclaving.
The PBST buffer method adopted in step (3) is as follows: NaCl8g, KCl0.2g, Na
2hPO
412H
2o2.9g, KH
2pO
40.2g, 500 μ L Tween-20s, supply DDW to 1L, regulate pH to 7.4, autoclaving.
Acetic acid-sodium acetate buffer solution formula in step (2) is as follows: sodium acetate 2.604g, glacial acetic acid 1.095g, supplies DDW to 1L, adjusts pH to 5.0, autoclaving.
Specific embodiment 3
Colloidal gold strip is to the detection of clinical sample, and concrete steps are as follows:
1, to the detection of tissue sample
Get the tissue sample of ill soft-shelled turtle, TNE damping fluid is dripped in organizing on sample, disrupting tissue, leave standstill after 5 minutes, test strip is inserted in measuring samples, observations after 10 minutes: only occur that a red line person is for positive in the quality control region of test strips, namely in sample with soft-shelled turtle systemic septicaemia spherical viruses; If ELISA test strip line and nature controlling line occur red line simultaneously, person is feminine gender.
2, to the detection of ascites sample
Get the ascites of ill soft-shelled turtle, test strip is inserted in soft-shelled turtle ascites sample to be checked, observations after 10 minutes.Criterion is with 1.
3, to the detection of hepatic pouch content supernatant
Get the hepatic pouch content of ill soft-shelled turtle, leave standstill and get supernatant, test strip is inserted in soft-shelled turtle hepatic pouch content Supernatant samples to be checked, observations after 10 minutes.Criterion is with 1.
4, to the detection of blood sample
Attack malicious infection experiment to healthy soft-shelled turtle, after attacking poison, 48h, 72h get its serum, test strip are inserted in soft-shelled turtle serum to be measured, observations after 10 minutes.Criterion is with 1.
5, to the detection of fecal specimens
Collect ill soft-shelled turtle ight soil, get supernatant with after TNE buffer solution fecal specimens, test strip is inserted in measuring samples, observations after 10 minutes.Criterion is with 1.
6, to the detection of ill turtle culture pond water
Get ill turtle culture pond Chi Shui, test strip is inserted in measuring samples, observations after 10 minutes.Criterion is with 1.
7, to the detection of negative sample
Test strip is inserted not containing in the DDW of soft-shelled turtle systemic septicaemia virus and in healthy soft-shelled turtle serum, observations after 10 minutes, have two band persons for negative.
8, all clinical sample testing result enzyme linked immunosorbent assays (ELISA) compare.
Testing result is in table 1, and we can see the tissue sample of ill soft-shelled turtle, ascites sample, hepatic pouch content Supernatant samples, and blood serum sample and fecal specimens all only have quality control region to occur a red line, and namely testing result is the positive.For not containing the DDW of soft-shelled turtle systemic septicaemia virus and the detection of healthy soft-shelled turtle blood sample, all having there is band in the detection line of result test strips and nature controlling line, is negative findings.All testing results all come to the same thing with ELISA, and both have high consistency.Infect the soft-shelled turtle serum display positive findings after 48h, show that we can carry out detection by colloidal gold strip at the soft-shelled turtle ill initial stage and assign a cause for an illness, thus suit the remedy to the case, timely control, prevent the state of an illness from expanding, reduce raiser's loss, simultaneously also can as a kind of preliminary screening instrument when seed is introduced.And detect ill soft-shelled turtle ight soil with colloidal gold strip and can assign a cause for an illness, convenient and practical especially, raiser only need collect the ight soil of soft-shelled turtle.
Table 1 soft-shelled turtle systemic septicaemia spherical viruses ELISA test strip clinical sample result
+: only there is nature controlling line red line in test strips, is positive findings;
-: there are two red lines in test strips, is negative findings.
Specific embodiment 4
The sensitivity technique of colloidal gold strip, concrete steps are as follows:
By the method for step (1) in specific embodiment 2, purifying soft-shelled turtle systemic septicaemia spherical viruses, carries out resuspended dilution with a small amount of PBS damping fluid, carries out measuring its protein content with ultramicron nucleic acid/protein assay (Nanodrop2000).Carried out a series of gradient dilution with PBS damping fluid, dilution is 5 μ g/mL respectively, 15 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL.Drawing soft-shelled turtle systemic septicaemia spherical viruses liquid (100 μ L) drips in the sample pad of test strip, leaves standstill observations after 10 minutes.With detection line disappear minimal detectable concentration be its lowest detection limit.
Testing result is in table 2, we can find out that in soft-shelled turtle systemic septicaemia spherical viruses concentration be 5 μ g/mL, 15 μ g/mL, test strips all occurs two lines during 25 μ g/mL, testing result is negative, and when soft-shelled turtle systemic septicaemia spherical viruses concentration is 50 μ g/mL, during 100 μ g/mL, detection line disappears, and only has control zone to occur a red line, and testing result is positive.Therefore, the detection limit of soft-shelled turtle systemic septicaemia spherical viruses test strip is 50 μ g/mL.
Table 2 soft-shelled turtle systemic septicaemia spherical viruses test strips sensitivity technique result
+: only there is nature controlling line red line in test strips, is positive findings;
-: there are two red lines in test strips, is negative findings.
Specific embodiment 5
The specific detection of soft-shelled turtle systemic septicaemia spherical viruses test strip, its concrete steps are as follows:
White spot syndrome virus (WSSV) (WSSV), Portunus trituberculatus Miers reovirus (CRV) and soft-shelled turtle systemic septicaemia spherical viruses (STSSSV) are diluted to 100 μ g/mL respectively, this three kinds of viruses are detected respectively by the test strips of preparation, often kind of virus does three repetitions, observations after 10 minutes.If test strips only occurs a red line in control zone, then testing result is positive, if the detection zone of test strips and control zone occur a red line all respectively, then testing result is negative.
In table 3, when we can find out with ELISA test strip prawn white spot syndrome (WSSV) and Portunus trituberculatus Miers reovirus (CRV), all there is a red line in the detection zone of test strips and control zone in testing result, namely testing result is negative respectively; And with only having occurred a red line in the control zone of test strips during ELISA test strip soft-shelled turtle systemic septicaemia spherical viruses, testing result is positive.As can be seen here, this soft-shelled turtle systemic septicaemia spherical viruses test strip has good specificity, and it with prawn white spot syndrome (WSSV) and Portunus trituberculatus Miers reovirus (CRV), cross reaction does not occur.
Table 3 soft-shelled turtle systemic septicaemia spherical viruses test strips specific detection result
。
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.Those skilled in the art are in essential scope of the present invention, and the change made, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (8)
1. a preparation method for soft-shelled turtle systemic septicaemia Viral diagnosis test strips, is characterized in that comprising the following steps:
(1) preparation of sample pad
All-glass paper is soaked in the PBS damping fluid containing the 0.01MpH7.4 of 1wt% bovine serum albumin(BSA), 2.5wt% sucrose, 1wt% trehalose, 1wt% Tween-20 and takes out after 30min, namely sample pad is obtained in 37 DEG C of dry 2-3h, Vacuum Package, 4 DEG C save backup;
(2) preparation of the pad of specified packet quilt
All-glass paper is soaked in containing 1wt% bovine serum albumin(BSA), 2.5wt% sucrose, 1wt% trehalose, in the PBS damping fluid of the 0.01MpH7.4 of 1wt% Tween-20 after 30min, after 37 DEG C of dry 1-2h, the anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing of even application 10-20 μ L on the all-glass paper of every square centimeter, vacuum freeze drying 1-2h, namely obtain pad, Vacuum Package, 4 DEG C save backup;
(3) preparation of the nitrocellulose filter of specified packet quilt
Soft-shelled turtle systemic septicaemia spherical viruses antigen point film instrument is coated on nitrocellulose membrane and is formed detection line; Be coated on nitrocellulose membrane by anti-for rabbit chicken yolk antibody point film instrument and form nature controlling line, namely obtain the nitrocellulose membrane of specified packet quilt, vacuum freeze drying 1-1.5h, Vacuum Package, 4 DEG C save backup; Wherein detection line and nature controlling line are parallel to each other, and described detection line is near pad end, and described nature controlling line is near adsorptive pads end;
(4) preparation of test strips
The nitrocellulose membrane of the specified packet quilt that the pad of the specified packet quilt that sample pad step (1) obtained, step (2) obtain, step (3) obtain and adsorptive pads overlap according to the order of sequence and are pasted on base plate, be cut into the slice that 4-6mm is wide, obtain soft-shelled turtle systemic septicaemia Viral diagnosis test strips, Vacuum Package, 4 DEG C of preservations.
2. the preparation method of a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips according to claim 1, it is characterized in that the preparation method of described soft-shelled turtle systemic septicaemia spherical viruses antigen is as follows: get and attack the little soft-shelled turtle that poison infects death, solution takes its internal organ on ice, weigh, by 1:5-1:20 add that ice bath in advance crosses containing homogenate in the TNE buffer solution of 1mM phenylmethylsulfonyl fluoride (PMSF), by homogenate in 4 DEG C, the centrifugal 20min of 8000g, precipitation with containing 1mMPMSF the homogenate of TNE damping fluid Eddy diffusion after again in 4 DEG C, the centrifugal 20min of 8000g, merge twice centrifugal gained supernatant, by supernatant in 4 DEG C, after the centrifugal 10min of 6000g, get supernatant in 4 DEG C, the centrifugal 1.5h of 38000g, wash away precipitation upper strata with the TM damping fluid of precooling to loosen part, take off layer solid white precipitate part with after resuspended containing the TM damping fluid of precooling of 1mMPMSF in 4 DEG C, after the centrifugal 5min of 3500g, get supernatant again in 4 DEG C, the centrifugal 1.5h of 38000g, wash away upper strata with TM damping fluid to loosen part, in 4 DEG C after the solid white precipitate of lower floor is again resuspended with the TM damping fluid of the precooling containing 1mMPMSF, after the centrifugal 5min of 3500g, get supernatant again in 4 DEG C, the centrifugal 1.5h of 38000g, wash away upper strata with TM damping fluid to loosen part, the solid white precipitate of the lower floor finally obtained is soft-shelled turtle systemic septicaemia spherical viruses antigen.
3. the preparation method of a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips according to claim 2, is characterized in that the preparation method of described anti-soft-shelled turtle systemic septicaemia virus Yolk antibody-colloid gold label thing is as follows:
(1) preparation of anti-soft-shelled turtle systemic septicaemia virus Yolk antibody
Get healthy first hen of laying eggs as immunization, by soft-shelled turtle systemic septicaemia viral antigen and the mixing of Freund's complete adjuvant equal-volume, the fully emulsified rear dipteron to hen, both legs, fundamental immunity is carried out in back and the intramuscular injection of chest muscle position, every hen immunizing dose is 500-800 μ g/mL, after 15-20 days, soft-shelled turtle systemic septicaemia viral antigen and incomplete Freund's adjuvant are carried out equal-volume mixing, fully emulsifiedly rear booster immunization is carried out to hen intramuscular injection, every hen immunizing dose is 400-600 μ g/mL, booster immunization repeats three times, each interval time is 7-10 days, then collect egg mensuration to tire, egg is collected when Yolk antibody tires >1:20000, be separated yolk, Yolk antibody is purified by saturated ammonium sulphate method,
(2) colloid gold label anti-soft-shelled turtle systemic septicaemia Yolk antibody
The colloidal gold solution being 15-30nm by anti-soft-shelled turtle systemic septicaemia virus Yolk antibody and colloid gold particle diameter mixes by 6-8 μ g:1mL, by stirring vibration 30-60min under the condition of pH5.4, add PBST damping fluid containing the bovine serum albumin(BSA) of 10wt% as stabilizing agent, adopt low-speed centrifugal 2000-3500rpm, 10-20min, high speed centrifugation 12000-15000rpm again, 1-1.5h, get kermesinus precipitation bottom centrifuge tube and namely obtain anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing, with containing 1wt% bovine serum albumin(BSA) by described anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody-colloid gold label thing, 2.5wt% sucrose, 1wt% trehalose, the resuspended 1/10-1/20 to described colloidal gold solution volume of 0.01MPBS damping fluid of the pH7.4 of 0.02wt% sodium azide is as its working concentration.
4. the preparation method of a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips according to claim 3, it is characterized in that purifying the concrete grammar of Yolk antibody by saturated ammonium sulphate method as follows: take appropriate step (1) and be separated the yolk obtained, 1:9 adds the acetic acid-sodium acetate buffer solution of the 0.05M of pH5.0 by volume, stir rear 4 DEG C of hold over night, the centrifugal 20min of 8000g, getting supernatant, to add saturated ammonium sulfate to saturation degree be 40%, 4 DEG C are stirred 6h, the centrifugal 20min of 10000g, the DDW of precipitation 10-20 times of yolk quality is resuspended, adding saturated sodium sulphate to saturation degree is 40%, 4 DEG C of stirrings are spent the night, the centrifugal 20min of 10000g, precipitate resuspended with the PBS damping fluid of the 0.01M of a small amount of pH7.4 after, in PBS damping fluid, namely dialysed overnight obtains anti-soft-shelled turtle systemic septicaemia spherical viruses Yolk antibody.
5. the preparation method of a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips according to claim 3, it is characterized in that in step (2), colloidal gold solution preparation method is as follows: by the chlorauric acid solution of 100mL0.01wt%, after heating is boiled, add 1-2mL1wt% trisodium citrate while stirring to shake up rapidly, continue heating, solution becomes claret by faint yellow blackening, heating 5-10min is continued to colour stable, dehydration is supplied to 100mL after cooling, sterile sealing, 4 DEG C keep in Dark Place.
6. the preparation method of a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips according to claim 3, is characterized in that: described detection line is apart from described pad 6-8mm; Described nature controlling line is apart from described adsorptive pads 6-8mm, and the width of described detection line and described nature controlling line is respectively 1-1.5mm, and two lines are apart 5mm.
7. the preparation method of a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips according to claim 3, it is characterized in that: described TNE buffer method is as follows: Tris6.0578g, NaCl12g, disodium ethylene diamine tetraacetate 1.8612g, supply DDW to 1L, regulate pH to 8.5, autoclaving;
Described TM buffer method is as follows: Tris6.0578g, MgCl
26H
2o2.0338g, supplies DDW to 1L, regulates pH to 7.4, autoclaving;
Described PBST buffer method is as follows: NaCl8g, KCl0.2g, Na
2hPO
412H
2o2.9g, KH
2pO
40.2g, 500 μ L Tween-20s, supply DDW to 1L, regulate pH to 7.4, autoclaving; Described PBS buffer method is as follows: NaCl8g, KCl0.2g, Na
2hPO
412H
2o2.9g, KH
2pO
40.2g, supplies DDW to 1L, regulates pH to 7.4, autoclaving;
Described acetic acid-sodium acetate buffer solution formula is as follows: sodium acetate 2.604g, glacial acetic acid 1.095g, supplies DDW to 1L, adjusts pH to 5.0, autoclaving.
8. the preparation method of a kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips according to claim 1, is characterized in that: the package amount of described soft-shelled turtle systemic septicaemia spherical viruses antigen is 2-4 μ L/cm; The package amount of the anti-chicken yolk antibody of described rabbit is 1-2 μ L/cm, and containing concentration in described soft-shelled turtle systemic septicaemia spherical viruses antigen is the phenylmethylsulfonyl fluoride of 1mM and the trehalose of 5wt%.
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