CN104730248A - Rapid diagnosis and detection test paper card for detecting swine fever as well as preparation and application method of rapid diagnosis and detection test paper card - Google Patents
Rapid diagnosis and detection test paper card for detecting swine fever as well as preparation and application method of rapid diagnosis and detection test paper card Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a rapid diagnosis and detection test paper card for detecting swine fever as well as a preparation and application method of the rapid diagnosis and detection test paper card. The rapid diagnosis and detection test paper card comprises a PVC base plate, a sample pad, a gold-labelled antibody glass fiber membrane, a nitrocellulose coating membrane, water absorption paper, a detection line and a quality control line. The rapid diagnosis and detection test paper card is applied based on a colloidal gold immunochromatography technology; in a detection process, a swine fever antigen in a sample is combined with a colloidal gold coated antibody to form an antigen-antibody complex, the antigen-antibody complex flows to the other end of an NC membrane along test paper, and when the complex flows to a detection line area on the membrane, a specific antibody fixed on the membrane can capture the complex and gradually coagulate the complex to form a visible detection line, an uncombined colloidal gold antibody flows through the detection line area and is captured by a second antibody of the quality control line to form a visible quality control line, and if the quality control line appears, the occurrence of immunochromatography can be determined, namely, the test paper is valid; and if the detection line appears, a swine fever virus can be determined to exist in the sample. The rapid diagnosis and detection test paper card disclosed by the invention can be used for judging results within 5 minutes, and has the characteristics of short diagnosis time and strong specificity.
Description
Technical field
The invention belongs to swine fever detection technique field, particularly relating to a kind of quick diagnosis Test paper card for detecting swine fever and preparation and application thereof.
Background technology
Swine fever is that the one caused by CSFV is acute, heat generation, contagious disease.China adopts immune hog cholera lapinised virus vaccine popular on a large scale in China of prevention and corntrol swine fever well, but in recent years swine fever have again stage a comeback, revivable sign, to be still one of Infectious Diseases of harm China pig industry at present.Pathogeny detection method because of its virus purification time long, the impact of the factors such as requirement condition is high, and separation rate is low, be difficult to mass detection is carried out to sample; And collected specimens carries out serological test, there is higher requirement to instrument and equipment and operating personnel, be not easy to the popularization in basic unit.Colloidal gold immunochromatographimethod skill is a kind of novel in vitro diagnostic techniques grown up the nineties in 20th century.This diagnostic method is fast and convenient, simple to operate, and without the need to instrument, result is accurate.Its ultimate principle is the osmosis concentration and the effect of capillary chromatography that utilize miillpore filter, and antigen-antibody is reacted on immobilon-p, and then develop the color with the antibody of colloid gold label, positive reaction presents redness on film, and negative reaction does not then occur redness.
At present, usually use swine fever reverse transcriptional PCR to carry out swine fever detection, concrete operation step is as follows:
1, sample collection: after animal dies of illness or slaughters, get tonsillotome, lymph node and spleen tissue ,-20 DEG C of refrigerations.
2, sample preparation: pathological material of disease of adopting fully grinds through tissue grinder, is collected in centrifuge tube by 1:5 physiological saline, and-70 DEG C of multigelations three times, 7000r/min, centrifugal 5 minutes, gets supernatant.
3, RNA extracts: the running program extracting kit according to Omega company RNA extracts total serum IgE.
4, RT-PCR running program: the single stage method RNA PCR kit running program according to TakaRa company is carried out.
Amplification condition is: 50 DEG C, reverse transcription 30 minutes, and 94 DEG C of sex change 5 minutes, enter circulation, 94 DEG C 50 seconds, 72 DEG C 60 seconds, latter 72 DEG C of 40 circulations extend 10 minutes.
5, RT-PCR product detects: amplified production is electrophoresis on 0.8% Ago-Gel, and EB dyes, and compares under ultraviolet light with standard molecular weight, and as seen 585bp fragment, tentative diagnosis is positive.
The shortcoming of prior art
In RT-PCR amplification procedure, need the Taq enzyme used, simultaneously because reasons such as the synthesis of test primer, cause experimentation cost higher; Organized processing step, RT-PCR amplification step cause test period long; The coloring agent EB (ethidium bromide) used in gel electrophoresis process has high carcinogenic.
Summary of the invention
The object of the present invention is to provide a kind of quick diagnosis Test paper card for detecting swine fever and preparation and application thereof, being intended to solve the problem that experimentation cost is higher, test period is long, coloring agent EB has high carcinogenic that RT-PCR amplification procedure exists.
The present invention realizes like this, for detecting a quick diagnosis Test paper card for swine fever, this quick diagnosis Test paper card being used for detecting swine fever comprises: PVC base plate, sample pad, golden labelled antibody glass fibre membrane, cellulose nitrate coated film, thieving paper, detection line, nature controlling line;
Apply the golden labelled antibody glass fibre membrane that every milliliter of golden labelled antibody solution applies the monoclonal antibody of the glass fibre element film colloid gold particle mark swine fever virus resistant of 10 square centimeters;
The bottom surface of cellulose nitrate coated film is pasted onto PVC substrate, difference sticking glass tunica fibrosa and the thieving paper above at cellulose nitrate coated film two ends, glass fibre membrane away from cellulose nitrate coated film one end above paste sample pad, detection line bag is provided with by the monoclonal antibody of swine fever virus resistant above cellulose nitrate coated film, nature controlling line bag is by anti-mouse antibody, cellulose nitrate coated film is nitrocellulose filter, and detection line bag is by the monoclonal antibody of swine fever virus resistant; The monoclonal antibody of swine fever virus resistant and anti-mouse antibody are individually fixed in also in two line parallels arrangements on cellulose nitrate coated film, and formation detection line, nature controlling line, undertaken qualitative by the colour developing of detection line and nature controlling line.
Another object of the present invention is to the preparation method providing a kind of quick diagnosis Test paper card for detecting swine fever, this preparation method being used for the quick diagnosis Test paper card detecting swine fever comprises the following steps:
Step one, the preparation of colloidal gold solution, puts distilled water on magnetic stirring apparatus and is heated with stirring to boiling, and the amount being 2/10000ths by final concentration adds rapidly the chlorauric acid solution that concentration is 1%, boils 5 minutes; Add by the volume of added chlorauric acid solution 1.8 times the citric acid three sodium solution that concentration is 1% again, after boiling 10 minutes, be cooled to room temperature, being settled to gold chloride final concentration with distilled water is 2/10000ths, and normal temperature keeps in Dark Place for subsequent use;
Step 2, the preparation of golden labeling antibody glass fibre membrane, regulates colloidal gold solution pH value to 7.5 with 5M sal tartari, adds the monoclonal antibody of swine fever virus resistant by the proportional concentration of 25 μ g antibody/milliliter colloidal gold solutions, and mixing leaves standstill 5 minutes; The BSA solution that concentration is 10% is added again by 5% volume, mixing, leave standstill 5 minutes, centrifugal 30 minutes of 10000rpm, abandons supernatant, precipitation mark cleansing solution washs once, abandon supernatant after centrifugal, the precipitation golden labeling antibody conserving liquid dissolving of 1/10th initial colloid gold volumes, the amount then spreading 10 square centimeters by every ml soln is coated on glass fibre membrane, vacuum drying, after 3 hours, is put in the sealing bag that drying agent is housed and is saved backup;
Step 3, the preparation of cellulose nitrate coated film, nitrocellulose filter sprays monoclonal antibody and the dynamics of swine fever virus resistant, and in two line parallel arrangements, form detection line and nature controlling line respectively, 37 DEG C of drying and processings 2 hours, put in the sealing bag that drying agent is housed and save backup;
Step 4, large plate group bar, each component specification (long × wide): PVC base plate: 30cm × 7.6cm; Sample pad: 30cm × 3.0cm; Gold labelled antibody glass fibre membrane: 30cm × 0.7cm; Cellulose nitrate coated film: 30cm × 2.0cm; Thieving paper: 30cm × 3.2cm;
Step 5, is cut into single person-portion with cutting cutter by large plate, and every person-portion width is 4 millimeters, inspects by random samples at random, and sensitivity can detect Internal Quality Control, and band colour developing degree reaches one "+" number, specific band nothing but;
Step 6,1 part of test strips cut, be responsible for a task until it is completed drying prescription and a plastic dropper are contained in aluminium foil bag, and with capper sealing, put into kit after sealing, air drying is preserved.
Further, in step 2, the concrete component proportion of 10%BSA solution, mark cleansing solution and golden labeling antibody conserving liquid is as follows:
10%BSA solution: containing bovine serum albumin(BSA) 100g in every 1000mL distilled water;
Mark cleansing solution: containing bovine serum albumin(BSA) 4g, 2g PEG 8000 in every 1000mL distilled water, polyvinylpyrrolidone 2g, sucrose 2g, Tris 1.211g, and regulate pH to 8.1;
Gold labeling antibody conserving liquid: containing bovine serum albumin(BSA) 25g, 5g PEG 8000 in every 1000mL distilled water, Tris 10.2g, NaOH 2.5g, 7.5mL Tween-20, and regulate pH to 8.5.
Further, in step 3:
Detection line, debugging spray film instrument, spouting liquid is 1 μ l/cm, and be buffered the monoclonal antibody of liquid dilution swine fever virus resistant with bag, concentration is 2.0mg/mL, with spray film instrument spraying line;
Nature controlling line, debugging spray film instrument, spouting liquid is 1 μ l/cm, and be buffered liquid dilution against murine IgG polyclonal antibody with bag, concentration is 2mg/mL, with spray film instrument spraying line.
Further, wrapping the concrete component proportion being buffered liquid is: containing bovine serum albumin(BSA) 0.01g, disodium hydrogen phosphate dodecahydrate 30.072g, potassium dihydrogen phosphate 2.176g in every 1000mL distilled water.
Further, in step 4, component carries out organizing bar as follows: be pasted onto the bottom surface of cellulose nitrate coated film on PVC base plate, on cellulose nitrate coated film two ends, paste golden labelled antibody glass fibre membrane and thieving paper respectively, paste sample pad at golden labelled antibody glass fibre membrane away from above one end of coated film; Namely on PVC base plate, mutually paste sample pad, golden labelled antibody glass fibre membrane, coated film and thieving paper in turn overlap joint, form large plate; The humidity of composing room will control below 30%.
Another object of the present invention is to the using method providing a kind of quick diagnosis Test paper card for detecting swine fever, this using method being used for the quick diagnosis Test paper card detecting swine fever comprises the following steps:
Step one, dips nasal secretion with cotton swab or gets blood testing;
Step 2, nasal secretion, immerses sample diluting liquid test tube by cotton swab, after stirring and evenly mixing, leave standstill, and gets supernatant as detection liquid with disposable dropper;
Step 3, serum, blood sampling 1ml, 1500 revs/min, centrifugal, get serum as detection liquid with disposable dropper;
Step 4, returns to room temperature by the test card do not broken a seal and detection sample;
Step 5, by test card horizontal positioned, dropwise adds 3 with dropper to test paper hole clipping and detects liquid;
Step 6,5 minutes judged results, the result after 10 minutes is invalid.
Quick diagnosis Test paper card for detecting swine fever provided by the invention and preparation and application thereof, the present invention is based on colloidal gold immunochromatographimethod technology, during detection, swine fever antigen in sample is combined with the antibody of collaurum bag quilt and forms antigen antibody complex, and the other end of NC film is flowed to along test paper, when this complex logistics is to detection line district on film, the specific antibody be fixed on film is caught this compound and is aggregated into a visible detection line gradually, unconjugated colloidal gold antibody flows through detection line district anti-caught by two of nature controlling line and forms visible nature controlling line, nature controlling line occurs then showing that immunochromatography occurs, namely test paper is effective, detection line line occurs then showing to contain CSFV in sample, within 5 minutes, gets final product result of determination, has Diagnostic Time short, the feature of high specificity.
Accompanying drawing explanation
Fig. 1 is the quick diagnosis Test paper card structure schematic diagram for detecting swine fever that the embodiment of the present invention provides;
In figure: 1, PVC base plate; 2, sample pad; 3, golden labelled antibody glass fibre membrane; 4, cellulose nitrate coated film; 5, thieving paper; 6, detection line; 7, nature controlling line.
Fig. 2 is the vertical view schematic diagram of Fig. 1 that the embodiment of the present invention provides;
Fig. 3 is the testing result that the embodiment of the present invention provides is the positive schematic diagram of CSFV;
Fig. 4 is the testing result that the embodiment of the present invention provides is negative schematic diagram;
Fig. 5 is the invalid schematic diagram of testing result that the embodiment of the present invention provides;
Fig. 6 is the using method process flow diagram of the quick diagnosis Test paper card for detecting swine fever that the embodiment of the present invention provides;
Fig. 7 is preparation method's process flow diagram of the quick diagnosis Test paper card for detecting swine fever that the embodiment of the present invention provides.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
As shown in Figure 1, mainly the comprising for the quick diagnosis Test paper card detecting swine fever of the embodiment of the present invention: PVC base plate 1, sample pad 2, golden labelled antibody glass fibre membrane 3, cellulose nitrate coated film 4, thieving paper 5, detection line 6, nature controlling line 7;
The golden labelled antibody glass fibre membrane 3 of the monoclonal antibody of PVC base plate 1, sample pad 2, coating (every milliliter of golden labelled antibody solution applies the glass fibre element film of 10 square centimeters) colloid gold particle mark (gold mark) swine fever virus resistant, coated film 4 and thieving paper 5;
The bottom surface of coated film 4 is pasted onto above PVC base plate 1, the sticking glass of the difference above tunica fibrosa 3 at cellulose nitrate coated film 4 two ends and thieving paper 5, glass fibre membrane 3 is away from the sample pad 2 of stickup above of one end of cellulose nitrate coated film 4, being provided with detection line 6 above cellulose nitrate coated film 4 wraps by the monoclonal antibody of swine fever virus resistant, nature controlling line 7 wraps by anti-mouse antibody, cellulose nitrate coated film 4 is nitrocellulose filter, and detection line 6 wraps by the monoclonal antibody of swine fever virus resistant; The monoclonal antibody of swine fever virus resistant and anti-mouse antibody are individually fixed in also in two line parallels arrangements on cellulose nitrate coated film 4, and formation detection line 6, nature controlling line 7, undertaken qualitative by the colour developing of detection line 6 and nature controlling line 7.
Principle of the present invention is: containing the monoclonal antibody of equally distributed colloid gold label swine fever virus resistant in test strips, the antibody of swine fever virus resistant and dynamics are separately fixed on nitrocellulose filter also in two rule parallel lines arrangements, against murine IgG is nature controlling line (negative control) up, is detection line under swine fever virus resistant antibody.
When containing CSFV in measuring samples, the swine fever virus resistant antibody that CSFV and gold mark is combined and forms antigenantibody complex, flow through from cellulose nitrate coated film with sample, swine fever virus resistant antibody on coated film combines with it, detection line forms red zone, and testing result is the CSFV positive (as shown in Figure 3).
When in testing sample during non-CSFV, the swine fever virus resistant antibody of gold mark cannot on cellulose nitrate coated film, swine fever virus resistant antibody is combined, cause the swine fever virus resistant antibody of gold mark cannot be detained on film, detection zone can not form red zone (i.e. detection line 6) testing result for negative (as shown in Figure 4).The testing result that quality control region forms this test strips of mark if any red zone is effective, otherwise the testing result of this test strips is invalid (as shown in Figure 5).
As shown in Figure 6, the using method for the quick diagnosis Test paper card detecting swine fever of the embodiment of the present invention comprises the following steps:
S601: dip nasal secretion with cotton swab or get blood testing;
S602: nasal secretion, immerses sample diluting liquid test tube by cotton swab, after fully stirring and evenly mixing, leave standstill a little time, gets supernatant as detection liquid with disposable dropper;
S603: serum, blood sampling 1ml, 1500 revs/min, centrifugal, get serum as detection liquid with disposable dropper;
S604: the test card do not broken a seal and detection sample are returned to room temperature;
S605: by test card horizontal positioned, slowly dropwise adds 3 with dropper to test paper hole clipping and detects liquid;
S606:5 minute judged result, the result after 10 minutes is invalid.
As shown in Figure 7, the preparation method for the quick diagnosis Test paper card detecting swine fever of the embodiment of the present invention comprises the following steps:
S701: the preparation of colloidal gold solution, puts distilled water on magnetic stirring apparatus and is heated with stirring to boiling, and the amount being 2/10000ths by final concentration adds rapidly the chlorauric acid solution that concentration is 1%, boils 5 minutes; Add by the volume of added chlorauric acid solution 1.8 times the citric acid three sodium solution that concentration is 1% again, after boiling 10 minutes, be cooled to room temperature, being settled to gold chloride final concentration with distilled water is 2/10000ths, and normal temperature keeps in Dark Place for subsequent use;
S702: the preparation of golden labeling antibody glass fibre membrane, regulates colloidal gold solution pH value to 7.5 with 5M sal tartari, adds the monoclonal antibody of swine fever virus resistant by the proportional concentration of 25 μ g antibody/milliliter colloidal gold solutions, and mixing leaves standstill 5 minutes; The BSA solution that concentration is 10% is added again by 5% volume, mixing, leave standstill 5 minutes, centrifugal 30 minutes of 10000rpm, abandons supernatant, precipitation mark cleansing solution washs once, abandon supernatant after centrifugal, the precipitation golden labeling antibody conserving liquid dissolving of 1/10th initial colloid gold volumes, the amount then spreading 10 square centimeters by every ml soln is coated on glass fibre membrane, vacuum drying, after 3 hours, is put in the sealing bag that drying agent is housed and is saved backup;
S703: the preparation of cellulose nitrate coated film, nitrocellulose filter sprays monoclonal antibody and the dynamics of swine fever virus resistant, and in two line parallel arrangements, form described detection line and nature controlling line respectively, 37 DEG C of drying and processings 2 hours, put in the sealing bag that drying agent is housed and save backup;
S704: large plate group bar, each component specification (long × wide): PVC base plate: 30cm × 7.6cm; Sample pad: 30cm × 3.0cm; Gold labelled antibody glass fibre membrane: 30cm × 0.7cm; Cellulose nitrate coated film: 30cm × 2.0cm; Thieving paper: 30cm × 3.2cm;
S705: large plate is cut into single person-portion with cutting cutter, every person-portion width is 4 millimeters, inspects by random samples at random, and sensitivity can detect Internal Quality Control, and band colour developing degree reaches one "+" number, specific band nothing but;
S706: 1 part of test strips cut, be responsible for a task until it is completed drying prescription and a plastic dropper are contained in aluminium foil bag, with capper sealing, put into kit after sealing, air drying is preserved.
In step S702, the concrete component proportion of above-mentioned 10%BSA solution, mark cleansing solution and golden labeling antibody conserving liquid is as follows:
10%BSA solution: containing bovine serum albumin(BSA) 100g in every 1000mL distilled water;
Mark cleansing solution: containing bovine serum albumin(BSA) 4g, 2g PEG 8000 in every 1000mL distilled water, polyvinylpyrrolidone 2g, sucrose 2g, Tris 1.211g, and regulate pH to 8.1;
Gold labeling antibody conserving liquid: containing bovine serum albumin(BSA) 25g, 5g PEG 8000 in every 1000mL distilled water, Tris 10.2g, NaOH 2.5g, 7.5mL Tween-20, and regulate pH to 8.5;
In step S703, detection line, debugging spray film instrument, spouting liquid is 1 μ l/cm, and be buffered the monoclonal antibody of liquid dilution swine fever virus resistant with bag, concentration is 2.0mg/mL, with spray film instrument spraying line;
Nature controlling line, debugging spray film instrument, spouting liquid is 1 μ l/cm, and be buffered liquid dilution against murine IgG polyclonal antibody with bag, concentration is 2mg/mL, with spray film instrument spraying line;
The concrete component proportion that above-mentioned bag is buffered liquid is: containing bovine serum albumin(BSA) 0.01g, disodium hydrogen phosphate dodecahydrate 30.072g, potassium dihydrogen phosphate 2.176g in every 1000mL distilled water;
On coated film draw two rule lines and answer fine uniform, interval 0.3cm;
In step S704, each component carries out organizing bar as follows: be pasted onto the bottom surface of cellulose nitrate coated film on PVC base plate, on these coated film two ends, paste golden labelled antibody glass fibre membrane and thieving paper respectively, paste sample pad at this golden labelled antibody glass fibre membrane away from above one end of coated film; Namely on PVC base plate, mutually paste sample pad, golden labelled antibody glass fibre membrane, coated film and thieving paper in turn overlap joint, form large plate; The humidity of composing room will control below 30%.
Colloidal gold immunochromatographimethod as the reliable immunology detection technology of one, high specificity, highly sensitive, easy fast, without the need to special instruments and equipment, be widely used value in animal quarantine real work.If strengthened up research and development, be expected the quick diagnosis and the detection that immune colloidal gold technique are widely used in various disease, for scientific, the standardization and high-speeding realizing China's animal quarantine work is significant.
Because collaurum has the observable redness of naked eyes, think without any need for the test paper film of instrument or the immune detection of RNA isolation kit and provide possibility, the pattern utilizing Immuno gold test paper embrane method to detect can be very flexible, for Pathogen test and medical diagnosis on disease provide a kind of detection means succinctly, easily and fast.
Living animal quarantine and the monitoring of the meat process of circulation are directly connected to the consumption safety of consumers in general and healthy, are the important contents of animal quarantine.And in the real work of China's animal quarantine, lack simple and effective detection means, the monitoring of living animal quarantine and the meat process of circulation rests on the original examination by sensory organs stage mostly, this situation constrains animal quarantine work efficiently and effectively and carries out, and also causes potential threat to China's animal husbandry safety in production simultaneously.Colloidal gold immunochromatographimethod technology has the plurality of advantages such as quick, sensitive, convenient, has larger realistic meaning for the quick diagnosis solving animal quarantine.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1. one kind for detecting the quick diagnosis Test paper card of swine fever, it is characterized in that, this quick diagnosis Test paper card being used for detecting swine fever comprises: PVC base plate, sample pad, golden labelled antibody glass fibre membrane, cellulose nitrate coated film, thieving paper, detection line, nature controlling line;
Apply the golden labelled antibody glass fibre membrane that every milliliter of golden labelled antibody solution applies the monoclonal antibody of the glass fibre element film colloid gold particle mark swine fever virus resistant of 10 square centimeters;
The bottom surface of cellulose nitrate coated film is pasted onto PVC substrate, difference sticking glass tunica fibrosa and the thieving paper above at cellulose nitrate coated film two ends, glass fibre membrane away from cellulose nitrate coated film one end above paste sample pad, detection line bag is provided with by the monoclonal antibody of swine fever virus resistant above cellulose nitrate coated film, nature controlling line bag is by anti-mouse antibody, cellulose nitrate coated film is nitrocellulose filter, and detection line bag is by the monoclonal antibody of swine fever virus resistant; The monoclonal antibody of swine fever virus resistant and anti-mouse antibody are individually fixed in also in two line parallels arrangements on cellulose nitrate coated film, and formation detection line, nature controlling line, undertaken qualitative by the colour developing of detection line and nature controlling line.
2. for detecting a preparation method for the quick diagnosis Test paper card of swine fever, it is characterized in that, this preparation method being used for the quick diagnosis Test paper card detecting swine fever comprises the following steps:
Step one, the preparation of colloidal gold solution, puts distilled water on magnetic stirring apparatus and is heated with stirring to boiling, and the amount being 2/10000ths by final concentration adds rapidly the chlorauric acid solution that concentration is 1%, boils 5 minutes; Add by the volume of added chlorauric acid solution 1.8 times the citric acid three sodium solution that concentration is 1% again, after boiling 10 minutes, be cooled to room temperature, being settled to gold chloride final concentration with distilled water is 2/10000ths, and normal temperature keeps in Dark Place for subsequent use;
Step 2, the preparation of golden labeling antibody glass fibre membrane, regulates colloidal gold solution pH value to 7.5 with 5M sal tartari, adds the monoclonal antibody of swine fever virus resistant by the proportional concentration of 25 μ g antibody/milliliter colloidal gold solutions, and mixing leaves standstill 5 minutes; The BSA solution that concentration is 10% is added again by 5% volume, mixing, leave standstill 5 minutes, centrifugal 30 minutes of 10000rpm, abandons supernatant, precipitation mark cleansing solution washs once, abandon supernatant after centrifugal, the precipitation golden labeling antibody conserving liquid dissolving of 1/10th initial colloid gold volumes, the amount then spreading 10 square centimeters by every ml soln is coated on glass fibre membrane, vacuum drying, after 3 hours, is put in the sealing bag that drying agent is housed and is saved backup;
Step 3, the preparation of cellulose nitrate coated film, nitrocellulose filter sprays monoclonal antibody and the dynamics of swine fever virus resistant, and in two line parallel arrangements, form detection line and nature controlling line respectively, 37 DEG C of drying and processings 2 hours, put in the sealing bag that drying agent is housed and save backup;
Step 4, large plate group bar, each component specification (long × wide): PVC base plate: 30cm × 7.6cm; Sample pad: 30cm × 3.0cm; Gold labelled antibody glass fibre membrane: 30cm × 0.7cm; Cellulose nitrate coated film: 30cm × 2.0cm; Thieving paper: 30cm × 3.2cm;
Step 5, is cut into single person-portion with cutting cutter by large plate, and every person-portion width is 4 millimeters, inspects by random samples at random, and sensitivity can detect Internal Quality Control, and band colour developing degree reaches one "+" number, specific band nothing but;
Step 6,1 part of test strips cut, be responsible for a task until it is completed drying prescription and a plastic dropper are contained in aluminium foil bag, and with capper sealing, put into kit after sealing, air drying is preserved.
3. as claimed in claim 2 for detecting the preparation method of the quick diagnosis Test paper card of swine fever, it is characterized in that, in step 2, the concrete component proportion of 10%BSA solution, mark cleansing solution and golden labeling antibody conserving liquid is as follows:
10%BSA solution: containing bovine serum albumin(BSA) 100g in every 1000mL distilled water;
Mark cleansing solution: containing bovine serum albumin(BSA) 4g, 2g PEG 8000 in every 1000mL distilled water, polyvinylpyrrolidone 2g, sucrose 2g, Tris 1.211g, and regulate pH to 8.1;
Gold labeling antibody conserving liquid: containing bovine serum albumin(BSA) 25g, 5g PEG 8000 in every 1000mL distilled water, Tris 10.2g, NaOH 2.5g, 7.5mL Tween-20, and regulate pH to 8.5.
4. as claimed in claim 2 for detecting the preparation method of the quick diagnosis Test paper card of swine fever, it is characterized in that, in step 3:
Detection line, debugging spray film instrument, spouting liquid is 1 μ l/cm, and be buffered the monoclonal antibody of liquid dilution swine fever virus resistant with bag, concentration is 2.0mg/mL, with spray film instrument spraying line;
Nature controlling line, debugging spray film instrument, spouting liquid is 1 μ l/cm, and be buffered liquid dilution against murine IgG polyclonal antibody with bag, concentration is 2mg/mL, with spray film instrument spraying line.
5. as claimed in claim 4 for detecting the preparation method of the quick diagnosis Test paper card of swine fever, it is characterized in that, the concrete component proportion that bag is buffered liquid is: containing bovine serum albumin(BSA) 0.01g in every 1000mL distilled water, disodium hydrogen phosphate dodecahydrate 30.072g, potassium dihydrogen phosphate 2.176g.
6. as claimed in claim 2 for detecting the preparation method of the quick diagnosis Test paper card of swine fever, it is characterized in that, in step 4, component carries out organizing bar as follows: be pasted onto the bottom surface of cellulose nitrate coated film on PVC base plate, on cellulose nitrate coated film two ends, paste golden labelled antibody glass fibre membrane and thieving paper respectively, paste sample pad at golden labelled antibody glass fibre membrane away from above one end of coated film; Namely on PVC base plate, mutually paste sample pad, golden labelled antibody glass fibre membrane, coated film and thieving paper in turn overlap joint, form large plate; The humidity of composing room will control below 30%.
7. for detecting a using method for the quick diagnosis Test paper card of swine fever, it is characterized in that, this using method being used for the quick diagnosis Test paper card detecting swine fever comprises the following steps:
Step one, dips nasal secretion with cotton swab or gets blood testing;
Step 2, nasal secretion, immerses sample diluting liquid test tube by cotton swab, after stirring and evenly mixing, leave standstill, and gets supernatant as detection liquid with disposable dropper;
Step 3, serum, blood sampling 1ml, 1500 revs/min, centrifugal, get serum as detection liquid with disposable dropper;
Step 4, returns to room temperature by the test card do not broken a seal and detection sample;
Step 5, by test card horizontal positioned, dropwise adds 3 with dropper to test paper hole clipping and detects liquid;
Step 6,5 minutes judged results, the result after 10 minutes is invalid.
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