CN1877331A - Foot-and-mouth disease virus detecting test paper tape and its preparation method and using method - Google Patents
Foot-and-mouth disease virus detecting test paper tape and its preparation method and using method Download PDFInfo
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- CN1877331A CN1877331A CN 200610043143 CN200610043143A CN1877331A CN 1877331 A CN1877331 A CN 1877331A CN 200610043143 CN200610043143 CN 200610043143 CN 200610043143 A CN200610043143 A CN 200610043143A CN 1877331 A CN1877331 A CN 1877331A
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Abstract
Disclosed are a method for preparing and virus type definition test paper for foot and mouth disease and the using method. The test paper comprises O, A, Asia l, C four kinds of serum detecting test paper. The test paper comprises a PVC substrate plate, nitrocellulose membrane, an absorbing pad, a golden mark pad and a sample pad. The PVC substrate plate is installed at the most bottom layer; the nitrocellulose membrane is set on the middle part of the substrate plate; the absorbing pad is stuck on the left end of the nitrocellulose membrane, and the golden mark pad is set on the right end of the nitrocellulose membrane; the sample pad is set on the right upper end of the golden pad. The invention can identify the foot and mouth virus O, A, Asia l and C four serum type.
Description
Technical field
The present invention relates to the serotype of foot and mouth disease virus is carried out diagnostic field quick and precisely, is a kind of foot-and-mouth disease virus detecting diagnosis test paper specifically, and the present invention includes the preparation and the using method of this test strips.
Background technology
Aftosa (Foot-and-mouth diease, FMD) be acute, hot, the contagious disease of suffering from altogether by the cloven-hoofed animal that foot and mouth disease virus (FMDV) causes, the height of its infection rate, velocity of propagation is fast, big to social danger, all occupy numerous eqpidemic diseases.International Office of Epizootics (OIE) classifies it first of category-A deadly infectious disease as.Because breaking out of aftosa had influence on the economic development of international relations, national reputation and countries in the world, therefore there is this disease of person to be " political economy disease ".Foot and mouth disease virus has O, A, C, Asia I, SATl, SAT2 and 7 serotypes of SAT3, more than 80 hypotype, no chiasma type immune response between serotype, and part cross immunity originality is only arranged between each hypotype, and foot and mouth disease virus constantly makes a variation, new variant and the continuous appearance that comes hypotype have brought great difficulty for the prevention and the control of aftosa.All take the measure strictly taken precautions against in each states of aspect such as international exchange, foreign trade, import and export, China also places the detection of hoof-and-mouth disease above the other things in animal and plant passed in and out quarantine law.
In May, 2005, State Council announced the popular situation of China Asia I type foot and mouth disease virus epidemic situation, and this makes epidemic prevention organizations at different levels pay attention to more aftosa diagnosis and prevention and control.Characteristics such as propagating when breaking out according to aftosa rapidly, break with tremendous force, the morbidity scope is wide, simple and rapid etiological diagnosis is particularly important to control epidemic situation.It can make a line epidemic prevention worker when epidemic situation is broken out correct diagnosis be made at the scene, in time determines cause of disease, cuts off the route of transmission, takes the effective precautionary measures to ensure health, the stable development of China's animal raising.
At present, the conventional method in detection of world aftosa reference laboratory foot and mouth disease virus and typing is still methods such as ELISA, virus separation, RT-PCR.These methods not only need certain instrument and equipment and laboratory facility, and need technician's operation of related experiment technical ability.The while complicated operation, process is various, and the cost height is unfavorable for carrying out of basic unit, field aftosa epidemiology survey and epidemic situation prevention and control.
Summary of the invention
The object of the present invention is to provide a kind of foot-and-mouth disease virus detecting diagnosis test paper of determining foot and mouth disease virus O, A, Asia I, four kinds of serotypes of C type.This test strips operation is fast and convenient, gets final product display result in the 15min, and it is directly perceived easy to judge, sensitivity is special as a result, and expense is cheap, and transportation is preserved convenient, is highly suitable for basic staff and detects use in the field.
Another object of the present invention is to provide the preparation method of this stereotype test strip.A further object of the present invention is to provide the using method of this stereotype test strip.
The objective of the invention is to realize by following technical solution:
A kind of aftosa stereotype test strip, form by O, A, four kinds of serotype test strip of Asia I, C, it is characterized in that described test strips partly is made up of PVC liner plate, nitrocellulose filter, absorption pad, gold mark pad, sample pad, described PVC liner plate is located at bottommost, stage casing, liner plate top is provided with nitrocellulose filter, nitrocellulose filter top left end posts absorption pad, and the nitrocellulose filter upper right end is provided with gold mark pad, and the upper right end of gold mark pad is provided with sample pad.
Described thieving paper and gold mark pad have overlapping with the joining place of nitrocellulose filter, between gold mark pad and the sample pad overlapping are arranged also.
To be sprayed with anti-rabbit, cavy and mouse IgG antibody be quality control band to left end on the described nitrocellulose filter; Right-hand member is sprayed with aftosa O, A, C, Asia I type antibody for detecting band on the nitrocellulose filter; Be sprayed with aftosa O, A, C, the Asia I type antibody of colloid gold label on the gold mark pad.
Aftosa O, the A, C, the Asia I type antibody that are sprayed with on described nitrocellulose filter and the gold mark pad are rabbit or cavy foot and mouth disease virus antibody or foot and mouth disease virus monoclonal antibody
The preparation method of above-mentioned aftosa stereotype test strip is:
1, the preparation of collaurum: adopt trisodium citrate reduction method to prepare collaurum;
2, the O of colloid gold label, A, C, Asia I type foot and mouth disease virus Antibody Preparation: at first a is gone on foot the collaurum 0.1mol/L K that makes
2CO
3The adjustment pH value is 8.0-8.5; Determine that with ocular estimate collaurum and antibody to be marked answers the optimised quantity of mark then, obtaining the optimum mark amount is 4-10mg/100mL, presses 4-10mg/100mL and add foot and mouth disease virus antibody in colloidal gold solution, stirs 10～20min; In colloidal gold solution, press 0.5-1g/100ml and add bovine serum albumin(BSA), continue to stir 10～20min, above-mentioned collaurum through the centrifugal 10-20 of 2000-4000r/min minute, is removed sediment, get supernatant; Supernatant is obtained sediment through 10000～12000r/min high speed centrifugation 1h, precipitation is suspended in the gold size damping fluid of the initial collaurum volume of 1/20-1/10,4 ℃ of preservations are put in the dissolving that suspends;
3, typing diagnosis test paper assembling:
3.1 preparation contains the nitrocellulose filter that detects band and quality control band: will resist rabbit, cavy and mouse IgGG antibody and O, A, C, Asia I foot-and-mouth disease antibody to be adjusted to 1-2mg/ml and 1.4-2.2mg/ml working concentration respectively with pH7.2-7.6 0.02mol/L phosphate buffer, by 1.8-2ul/cm the spray film is set respectively, above-mentioned two kinds of antibody are sprayed on the nitrocellulose filter, form quality control band and detect band, 37 ℃ of oven dry or after room temperature dries, place confining liquid to soak 5-15min, take out under the room temperature of back and dry, preserve standby;
3.2 preparation collaurum pad: get all-glass paper, by 10-20 μ L/cm O, A, C, the Asia I foot-and-mouth disease antibody of colloid gold label are sprayed at respectively on the all-glass paper, dry back is stored standby;
3.3 the assembling of test strips: at the PVC backer board from the bottom to top respectively with sample pad, collaurum pad, comprise that the nitrocellulose filter and the absorption pad that detect band, quality control band assemble in order, cut into strip, promptly make different shaped foot and mouth disease virus diagnosis test paper, aftosa O, A, C, four kinds of test strips of Asia I type are packed into as a packing and are aftosa typing diagnosis test paper in the aluminium plastic bag.
Above-mentioned gold size damping fluid is the phosphate buffer that contains the PH7.5 0.11mol/L of 5-15% sucrose, 1%BSA, 0.5-1 ‰ PEG20000,0.5-1 ‰ Tween-20.
Above-mentioned confining liquid is for containing the 2.5-5% calf serum, the phosphate buffer of the PH7.4 0.02mol/L of 0.5-1 ‰ Tween-20.
The using method of aftosa stereotype test strip of the present invention is:
1, the processing of sample to be checked: sterile working is washed blister skin to be checked or suckling mouse tissue 2-3 time with the phosphate buffer of PH7.2-7.50.02-0.05mol/L, and inhale the part of anhydrating with sterilized filter paper and weigh, adding glass sand grinds, by 1: 3～1: 10w/v adds the phosphate buffer of PH7.2-7.5 0.02-0.05mol/L, makes suspension; Room temperature is soaked in poison 2 hours or 4 ℃ of refrigerators and is soaked a malicious night.With the centrifugal 10min of 4000r/min, get supernatant and be sample to be checked after the jolting; Blister liquid to be checked can be directly as sample to be checked;
2, sample detection: sample to be checked, detect test paper or other and detect with material etc., have an end of markings to insert in the sample to be checked detecting test paper all at equilibrium at room temperature, after liquid all soaks nitrocellulose filter, observations in 5-15 minute;
3, the result judges
3.1 single test strips result judges:
Positive: quality control band (6) high-visible red stripes all occurs with detection band (7);
Negative: as to have only quality control band (6) high-visible red stripes to occur;
Suspicious: the apparent red stripes of color appears in quality control band (6), and is detecting light color band of band (7) appearance;
Invalid: red stripes does not appear in quality control band (6);
3.2 typing is the result judge:
The O type: the result is positive for O type test strips, and the result is negative or suspicious for Asia I, A, C type test strips;
Asia I type: the result is positive for Asia I type test strips, and the result is negative or suspicious for O, A, C type test strips.
The A type: the result is positive for A type test strips, and the result is negative or suspicious for O, Asia I, C type test strips.
The C type: the result is positive for C type test strips, and the result is negative or suspicious for O, Asia I, A type test strips.
Negative: the result is all negative for O, Asia I, A, C type test strips.
Suspicious: O, Asia I, A, C type test strips result are suspicious, or wherein three kinds, two kinds test strips results are suspicious.
When four kinds, three kinds and two kinds of test strips are all positive to same antigen colour developing result to be checked, sample to be checked can be the two-fold dilution with dilution, detect again, judge c.2 to carry out the result;
Above-mentioned dilution is the PH7.5 0.11Mol/L phosphate buffer that contains 0.5-1 ‰ Tween-20.
The colloidal gold immunochromatographimethod technology (A gold immunochromatographic assayGICA) that the present invention adopts is a kind of solid phase labelling immunoassay technology that several different methods such as colloidal gold-labeled method, immunoassay technology and chromatographic analysis technology are organically combined.It is to be solid phase with the fibre strip chromatographic material, make sample solution swimming on chromatography strip by capillary action, and make the immune response that high specific, high-affinity take place at the acceptor (antigen or antibody) of determinand on determinand in the sample and the chromatographic material simultaneously, immune complex is by enrichment or be trapped in certain zone (detect band) of chromatographic material in the chromatography process, the label that utilization can be estimated (collaurum) and obtain experimental phenomena (colour developing) intuitively.Free label is then crossed and is detected band, separates automatically with binding label.This analytical technology has the instrument and equipment that fast, do not need simple to operate; It is directly perceived, sensitive and accurate that the result judges; Objectionable impuritiess such as "dead" isotope and phenylenediamine participate in, and do not pollute the environment, and security is good; Can single part of mensuration, required reagent and sample size are few, and be with low cost; Preserve and transport advantages such as simple and convenient.
Therefore, concerning the zymad aftosa of state key prevention and control, utilize the new fast and convenient diagnostic techniques of GICA development, can be used as the useful of Routine Test Lab diagnostic method on the one hand and replenish, will provide great convenience for aftosa typing diagnosis grass-roots work personnel on the other hand.
The evaluation experimental example:
1. susceptibility experiment
Getting the positive sample that national aftosa reference laboratory has been diagnosed as aftosa O, A, C, Asia I serotype detects with stereotype test strip for 50 parts.Coincidence rate is 92%.
2. specificity experiment
Detect bubble skin, BF and the normal suckling mouse tissue samples that national aftosa reference laboratory has been diagnosed as the aftosa feminine gender with stereotype test strip and amount to 20 parts.Detecting coincidence rate is 100%.
3. repeated experiment
Get different production batch 041122,050310,050815 test strips respectively the above-mentioned positive and negative sample are carried out duplicate detection, the coincidence rate of three detections is 100%.
4. stability experiment
Test strips is placed on respectively under the different condition stores: 37 ℃ of one week, 4 ℃ 1 year, took out with January every 1 day the identical positive and negative sample are tested.All have the quality control band that shows Success in Experiment to occur during test, to the detection of positive, quality control band and detection band colour developing degree and developing time there was no significant difference illustrate its stability better simultaneously.
5. the detection of field sample
Use test strips the sample and the separation and Culture thing of inspecting by ready samples detected, comprise that cell toxicant, sodoku, bubble skin, BF, O/P liquid etc. amount to 107 duplicate samples and carry out parallel contrast experiment with reverse indirect hemagglutination respectively.The coincidence rate that gold test strip and reverse indirect blood coagulation detect is 93.61%, there was no significant difference.
The invention has the advantages that:
1, easy and simple to handle, it is directly perceived easy to judge, does not need any instrument and equipment.General personnel get final product complete operation to specifications, do not need special training.、
2, method of testing is fast rapid, and whole test process only needed to finish in 15 minutes.
3, manufacture with low costly, required reagent and sample size are few.
4, transportation is preserved conveniently.Because colloid gold label protein is a physical bond process, in conjunction with firmly, seldom cause the protein active change, so the test strips steady quality is not influenced by extraneous factor such as temperature, can transport preservation at room temperature.
5, more environmental protection of safety non-pollution.Do not participate in such as objectionable impuritiess such as radioactive isotope, o-phenylenediamines, thus do not pollute the environment yet, have detection methods such as radioactive isotope or enzyme mark incomparable security.
6, the result is sensitive and accurate, specificity good, and it is less that the result is influenced by extraneous factor.
Can predict by above-mentioned advantage, in the rural area, field, basic unit, to the FMDV epidemic situation follow the tracks of and epidemiology survey work in, this invention will possess bigger application prospect.
Description of drawings
Fig. 1 is a test strips structural representation of the present invention
Fig. 2 is a colloid gold particle transmission electron microscope synoptic diagram
Fig. 3 is a test strips positive findings synoptic diagram
Fig. 4 is a test strips negative findings synoptic diagram
Fig. 5 is the suspicious result schematic diagram of test strips
Fig. 6 is for detecting the test strips null result synoptic diagram of band colour developing
Fig. 7 is for detecting the test strips null result synoptic diagram that band does not develop the color
Embodiment
Below in conjunction with embodiment the present invention is further elaborated.
Embodiment 1
The preparation of O type aftosa diagnosis test paper
1, the preparation of collaurum
Use trisodium citrate reduction method, getting 0.01% aqueous solution of chloraurate 100mL is heated to and boils, stir and accurately add 1% trisodium citrate aqueous solution 2mL down, flavous aqueous solution of chloraurate becomes aubergine in 2min, continue to boil 15min, cooling back returns to original volume with distilled water, and so the highest absorption peak of its visible region of aurosol of preparation is at 523nm, A1cm/523=1.188.The NaN of adding 0.02% in the collaurum of preparation
3, filter through the primary sterilization filter, standby in the clean glass bottle of packing into.Collaurum diameter prepared in the work is by observing grain size at transmission electron microscope (TEM picture).Recording mean grain size is 30.06 ± 0.7nm, and the result as shown in Figure 2.
2, the preparation of immune colloid gold
Use 0.1mol/L K
2CO
3Adjusting collaurum colloidal sol extremely required pH is 8.2, adds O type aftosa cavy IgG, magnetic agitation 15min by 45 μ g/mL collaurums.In colloidal gold solution, press 1% and add bovine serum albumin(BSA), continue to stir 15min, above-mentioned collaurum was removed sediment in centrifugal 15 minutes through .3000r/min, get supernatant; Supernatant is obtained sediment through 10000r/min high speed centrifugation 1h, precipitation is suspended in the PH7.50.11mol/LPBS gold size damping fluid that contains 10% sucrose, 1%BSA, 1 ‰ PEG20000,1 ‰ Tween-20 of 8/100 initial collaurum volume, suspend and dissolve, put 4 ℃ of preservations.
3, the preparation method of aftosa typing diagnosis test paper
3.1 preparation contains the nitrocellulose filter that detects band and quality control band
Respectively the O type FMDV rabbit igg of purifying being adjusted to concentration with pH7.2 0.02mol/LPBS is 2.2mg/mL, and the anti-cavy IgG of rabbit concentration is 1.0mg/mL.By 1.8ul/cm the spray film is set respectively, two kinds of IgG is sprayed on the NC film of 300mm * 20mm, form and detect band and quality control band, two lines place the PH7.2 0.02mol/LPBS coating buffer that contains 2.5% calf serum, 1 ‰ Tween-20 to soak 5min at a distance of 8mm.Take out back 37 ℃ of oven dry 1h, preserve standby.
3.2 preparation collaurum pad
Get all-glass paper, by 15 μ L/cm, the O type FMDV cavy IgG with colloid gold label is sprayed on the all-glass paper with Membrane jetter, and dry back is stored standby.
3.3 the assembling of test strips
To draw the absorption of sample pad that sample uses, the collaurum pad that is fixed with colloid gold label O type foot-and-mouth disease antibody respectively at the PVC backer board, wrap the nitrocellulose filter of tested measuring tape, quality control band and absorption pad that absorbent filter is made by from the bottom to top by diagram 1 assembling, thieving paper and gold mark pad must have overlapping 0.1cm with the nitrocellulose filter film, and gold mark pad is also used overlapping 0.1cm with sample pad.Accessory is bonding with the available double faced adhesive tape of combining of plastic bottom board or other cohesive material.The cardboard that assembles is cut into the strip that width is 2.5mm by longitudinal shear, promptly makes O type FMDV diagnosis test paper.
The preparation of A type aftosa diagnosis test paper
1, the preparation of collaurum
Use trisodium citrate reduction method, getting 0.01% aqueous solution of chloraurate 100mL is heated to and boils, stir and accurately add 1% trisodium citrate aqueous solution 2mL down, flavous aqueous solution of chloraurate becomes aubergine in 2min, continue to boil 15min, cooling back returns to original volume with distilled water, and so the highest absorption peak of its visible region of aurosol of preparation is at 523nm, A1cm/523=1.188.The NaN of adding 0.02% in the collaurum of preparation
3, filter through the primary sterilization filter, standby in the clean glass bottle of packing into.Collaurum diameter prepared in the work is by observing grain size at transmission electron microscope (TEM picture).Recording mean grain size is 30.06 ± 0.7nm, and the result as shown in Figure 2.
2, the preparation of immune colloid gold
Use 0.1mol/L K
2CO
3Adjusting collaurum colloidal sol extremely required pH is 8.0, adds A type aftosa rabbit igg, magnetic agitation 10min by 40 μ g/mL collaurums.In colloidal gold solution, press 0.5% and add bovine serum albumin(BSA), continue to stir 10min, above-mentioned collaurum was removed sediment in centrifugal 10 minutes through 2000r/min, get supernatant; Supernatant is obtained sediment through 10000r/min high speed centrifugation 1h, precipitation is suspended in the PH7.40.11mol/LPBS gold size damping fluid that contains 5% sucrose, 0.5%BSA, 0.5 ‰ PEG20000,0.5 ‰ Tween-20 of 1/20 initial collaurum volume, suspend and dissolve, put 4 ℃ of preservations.
3, the preparation method of aftosa typing diagnosis test paper
3.1 preparation contains the nitrocellulose filter that detects band and quality control band
Respectively the A type FMDV cavy IgG of purifying being adjusted to concentration with pH7.2 0.02mol/LPBS is 1.4mg/mL, and goat anti-rabbit igg concentration is 1.0mg/mL.By 2ul/cm the spray film is set respectively, two kinds of IgG is sprayed on the NC film of 300mm * 20mm, form and detect band and quality control band, two lines place the PH7.4 0.02mol/L PBS coating buffer that contains 2.59% calf serum, 0.5 ‰ Tween-20 to soak 5min at a distance of 8mm.Take out back 37 ℃ of oven dry 1h, preserve standby.
3.2 preparation collaurum pad
Get all-glass paper, by 10 μ L/cm, the A type FMDV rabbit igg with colloid gold label is sprayed on the all-glass paper with Membrane jetter, and dry back is stored standby.
3.3 the assembling of test strips
To draw the absorption of sample pad that sample uses, the collaurum pad that is fixed with colloid gold label A type foot-and-mouth disease antibody respectively at the PVC backer board, wrap the nitrocellulose filter of tested measuring tape, quality control band and absorption pad that absorbent filter is made by from the bottom to top by diagram 1 assembling, thieving paper and gold mark pad must have overlapping 0.1cm with the nitrocellulose filter film, and gold mark pad is also used overlapping 0.1cm with sample pad.Accessory is bonding with the available double faced adhesive tape of combining of plastic bottom board or other cohesive material.The cardboard that assembles is cut into the strip that width is 2.5mm by longitudinal shear, promptly makes A type FMDV diagnosis test paper.
The preparation of C type aftosa diagnosis test paper
1, the preparation of collaurum
Use trisodium citrate reduction method, getting 0.01% aqueous solution of chloraurate 100mL is heated to and boils, stir and accurately add 1% trisodium citrate aqueous solution 2mL down, flavous aqueous solution of chloraurate becomes aubergine in 2min, continue to boil 15min, cooling back returns to original volume with distilled water, and so the highest absorption peak of its visible region of aurosol of preparation is at 523nm, A1cm/523=1.188.The NaN of adding 0.02% in the collaurum of preparation
3, filter through the primary sterilization filter, standby in the clean glass bottle of packing into.Collaurum diameter prepared in the work is by observing grain size at transmission electron microscope (TEM picture).Recording mean grain size is 30.06 ± 0.7nm, and the result as shown in Figure 2.
2, the preparation of immune colloid gold
Use 0.1mol/L K
2CO
3Adjusting collaurum colloidal sol extremely required pH is 8.5, adds C type foot and mouth disease virus monoclonal antibody, magnetic agitation 20min by 100 μ g/mL collaurums.In colloidal gold solution, press 1% and add bovine serum albumin(BSA), continue to stir 20min, above-mentioned collaurum was removed sediment in centrifugal 20 minutes through 4000r/min, get supernatant; Supernatant is obtained sediment through 12000r/min high speed centrifugation 1h,. precipitation is suspended in the PH7.6 0.11mol/L PBS gold size damping fluid that contains 15% sucrose, 1%BSA, 1 ‰ PEG20000,1 ‰ Tween-20 of 1/10 initial collaurum volume, suspend and dissolve, put 4 ℃ of preservations.
3, the preparation method of aftosa typing diagnosis test paper
3.1 preparation contains the nitrocellulose filter that detects band and quality control band
Respectively the C type FMDV cavy IgG of purifying being adjusted to concentration with pH7.2 0.02mol/LPBS is 2.2mg/mL, and rabbit anti-mouse igg concentration is 2.0mg/mL.By 1.8ul/cm the spray film is set respectively, two kinds of IgG is sprayed on the NC film of 300mm * 20mm, form and detect band and quality control band, two lines place the PH7.6 0.02mol/LPBS coating buffer that contains 5% calf serum, 1 ‰ Tween-20 to soak 15min at a distance of 8mm.Take out back 37 ℃ of oven dry 1h, preserve standby.
3.2 preparation collaurum pad
Get all-glass paper, by 10 μ L/cm, the C type foot and mouth disease virus monoclonal antibody with colloid gold label is sprayed on the all-glass paper with Membrane jetter, and dry back is stored standby.
3.3 the assembling of test strips
To draw the absorption of sample pad that sample uses, the collaurum pad that is fixed with colloid gold label C type foot-and-mouth disease antibody respectively at the PVC backer board, wrap the nitrocellulose filter of tested measuring tape, quality control band and absorption pad that absorbent filter is made by from the bottom to top by diagram 1 assembling, thieving paper and gold mark pad must have overlapping 0.1cm with the nitrocellulose filter film, and gold mark pad is also used overlapping 0.1cm with sample pad.Accessory is bonding with the available double faced adhesive tape of combining of plastic bottom board or other cohesive material.The cardboard that assembles is cut into the strip that width is 2.5mm by longitudinal shear, promptly makes C type FMDV diagnosis test paper.
The preparation of Asia I type aftosa diagnosis test paper
1, the preparation of collaurum
Use trisodium citrate reduction method, getting 0.01% aqueous solution of chloraurate 100mL is heated to and boils, stir and accurately add 1% trisodium citrate aqueous solution 2mL down, flavous aqueous solution of chloraurate becomes aubergine in 2min, continue to boil 15min, cooling back returns to original volume with distilled water, and so the highest absorption peak of its visible region of aurosol of preparation is at 523nm, A1cm/523=1.188.The NaN of adding 0.02% in the collaurum of preparation
3, filter through the primary sterilization filter, standby in the clean glass bottle of packing into.Collaurum diameter prepared in the work is by observing grain size at transmission electron microscope (TEM picture).Recording mean grain size is 30.06 ± 0.7nm, and the result as shown in Figure 2.
2, the preparation of immune colloid gold
Use 0.1mol/L K
2CO
3Adjusting collaurum colloidal sol extremely required pH is 8.4, adds Asia I type FMDV rabbit igg, magnetic agitation 20min by 60 μ g/mL collaurums.In colloidal gold solution, press 1% and add bovine serum albumin(BSA), continue to stir 20min, above-mentioned collaurum was removed sediment in centrifugal 15 minutes through 3000r/min, get supernatant; Supernatant is obtained sediment through 11000r/min high speed centrifugation 1h, precipitation is suspended in the PH7.5 0.11mol/LPBS gold size damping fluid that contains 10% sucrose, 1%BSA, 0.5 ‰ PEG20000,0.5 ‰ Tween-20 of 8/100 initial collaurum volume, suspend and dissolve, put 4 ℃ of preservations.
3, the preparation method of aftosa typing diagnosis test paper
3.1 preparation contains the nitrocellulose filter that detects band and quality control band
Respectively the Asia I type foot and mouth disease virus monoclonal antibody of purifying being adjusted to concentration with pH7.5 0.02mol/LPBS is 1.8mg/mL, and goat anti-rabbit igg concentration is 1.5mg/mL.By 2ul/cm the spray film is set respectively, two kinds of IgG is sprayed on the NC film of 300mm * 20mm, form and detect band and quality control band, two lines place the PH7.5 0.02mol/L PBS coating buffer that contains 5% calf serum, 1 ‰ Tween-20 to soak 15min at a distance of 8mm.Take out back 37 ℃ of oven dry 1h, preserve standby.
3.2 preparation collaurum pad
Get all-glass paper, by 15 μ L/cm, the Asia I type FMDV rabbit igg with colloid gold label is sprayed on the all-glass paper with Membrane jetter, and dry back is stored standby.
3.3 the assembling of test strips
To draw the absorption of sample pad that sample uses, the collaurum pad that is fixed with colloid gold label Asia I type foot-and-mouth disease antibody respectively at the PVC backer board, wrap the nitrocellulose filter of tested measuring tape, quality control band and absorption pad that absorbent filter is made by from the bottom to top by diagram 1 assembling, thieving paper and gold mark pad must have overlapping 0.1cm with the nitrocellulose filter film, and gold mark pad is also used overlapping 0.1cm with sample pad.Accessory is bonding with the available double faced adhesive tape of combining of plastic bottom board or other cohesive material.The cardboard that assembles is cut into the strip that width is 2.5mm by longitudinal shear, promptly makes Asia I type FMDV diagnosis test paper.
Foot-and-mouth disease virus detecting diagnostic test method
1. the processing of sample to be checked: sterile working is washed blister skin to be checked (taking fresh end to break, do not have the bubble skin of peculiar smell) 2 times with PH7.5 0.05mol/L PBS damping fluid, and inhale the part of anhydrating with sterilized filter paper and weigh, adding glass sand grinds, make suspension by 1: 10 (w/v) PH7.5 0.05mol/LPBS damping fluid, soak a malicious night in 4 ℃ of refrigerators.With the centrifugal 10min of 4000r/min, get supernatant and be sample to be checked after the jolting.Can be directly as sample to be checked.
2. sample detection: sample to be checked, detect test paper or other and detect with material etc. all at equilibrium at room temperature.Tear along aluminium bag cutting part, take out test strips.There is an end of markings to insert in the sample to be checked with detecting test paper, after liquid all soaks nitrocellulose filter, observations in 5 minutes.
3. the result judges
3.1 single test strips result judges:
Positive: quality control band (6) high-visible red stripes all occurs with detection band (7).Foot and mouth disease virus content in the sample is high more, detects and is with color dark more redly.See diagram 3
Negative: as to have only quality control band (6) high-visible red stripes to occur.See diagram 4
If suspicious: the apparent red line of color appears in quality control band (6), and is detecting very slight color of band (7) appearance, if latent the red zone that shows.See diagram 5
Invalid: red stripes does not appear in quality control band (6).See diagram 6, diagram 7
3.2 typing is the result judge:
The O type: the result is positive for O type test strips, and the result is negative or suspicious for Asia I, A, C type test strips.
Asia I type: the result is positive for AsiaI type test strips, and the result is negative or suspicious for O, A, C type test strips.
The A type: the result is positive for A type test strips, and the result is negative or suspicious for Asia I, O, C type test strips.
The C type: the result is positive for C type test strips, and the result is negative or suspicious for Asia I, O, A type test strips.
Negative: the result is all negative for O, Asia I, A, C type test strips.
Suspicious: O, Asia I, A, C type test strips result are suspicious, or wherein three kinds, two kinds test strips results are suspicious.
Annotate: when four kinds, three kinds and two kinds of test strips are all positive to same antigen colour developing result to be checked, sample to be checked can be the two-fold dilution with the PH7.4 0.11MPBS that contains 0.5 ‰ Tween-20, detect again, carry out the result with the same method and judge.
Foot-and-mouth disease virus detecting diagnostic test method
1, the processing of sample to be checked: sterile working is washed the suckling mouse tissue 3 times with the 0.05mol/L phosphate buffer of PH7.2, and inhale the part of anhydrating with sterilized filter paper and weigh, add glass sand and grind, by 1: 3w/v adds the phosphate buffer of PH7.2 0.05mol/L, makes suspension; Room temperature was soaked poison 2 hours.With the centrifugal 10min of 4000r/min, get supernatant and be sample to be checked after the jolting; Blister liquid to be checked can be directly as sample to be checked;
2, sample detection: sample to be checked, detect test paper or other and detect with material etc., have an end of markings to insert in the sample to be checked detecting test paper all at equilibrium at room temperature, after liquid all soaks nitrocellulose filter, observations in 15 minutes;
3, the result judges
3.1 single test strips result judges:
Positive: quality control band (6) high-visible red stripes all occurs with detection band (7);
Negative: as to have only quality control band (6) high-visible red stripes to occur;
Suspicious: the apparent red stripes of color appears in quality control band (6), and is detecting light color band of band (7) appearance;
Invalid: red stripes does not appear in quality control band (6);
3.2 typing is the result judge:
The O type: the result is positive for O type test strips, and the result is negative or suspicious for Asia I, A, C type test strips;
Asia I type: the result is positive for Asia I type test strips, and the result is negative or suspicious for O, A, C type test strips.
The A type: the result is positive for A type test strips, and the result is negative or suspicious for O, Asia I, C type test strips.
The C type: the result is positive for C type test strips, and the result is negative or suspicious for O, Asia I, A type test strips.
Negative: the result is all negative for O, Asia I, A, C type test strips.
Suspicious: O, Asia I, A, C type test strips result are suspicious, or wherein three kinds, two kinds test strips results are suspicious.
When four kinds, three kinds and two kinds of test strips are all positive to same antigen colour developing result to be checked, sample to be checked can be the two-fold dilution with dilution, detect again, judge c.2 to carry out the result;
Above-mentioned dilution is the PH7.6 0.11Mol/L phosphate buffer that contains 1 ‰ Tween-20s.
Claims (10)
1 one kinds of aftosa stereotype test strips, form by O, A, four kinds of serotype test strip of Asia I, C, it is characterized in that described test strip is made up of PVC liner plate, nitrocellulose filter, absorption pad, gold mark pad, sample pad 5 parts, described PVC liner plate (1) is located at bottommost, liner plate (1) stage casing, top is provided with nitrocellulose filter (2), nitrocellulose filter (2) top left end posts absorption pad (3), nitrocellulose filter (2) upper right end is provided with gold mark pad (4), and the upper right end of gold mark pad (4) is provided with sample pad (5).
2, aftosa stereotype test strip according to claim 1 is characterized in that described thieving paper (3) and gold mark pad (4) and the joining place of nitrocellulose filter (2) have overlapping, also have overlapping between gold mark pad (4) and the sample pad (5).
3, aftosa stereotype test strip according to claim 1 is characterized in that the last left end of described nitrocellulose filter (2) is sprayed with anti-rabbit, cavy and mouse IgG antibody as quality control band (6); Nitrocellulose filter (2) is gone up right-hand member and is sprayed with aftosa O, A, C, Asia I type antibody as detecting band (7); Be sprayed with aftosa O, A, C, the Asia I type antibody of colloid gold label on the gold mark pad (4).
4, aftosa stereotype test strip according to claim 3 is characterized in that the colloid gold label aftosa O, A, C, the Asia I type antibody that are sprayed with on the described gold mark pad (4) are rabbit or cavy foot and mouth disease virus antibody or foot and mouth disease virus monoclonal antibody.
5, aftosa stereotype test strip according to claim 3 is characterized in that described detection band (7) is sprayed with aftosa O, A, C, Asia I type antibody is rabbit or cavy foot and mouth disease virus antibody or foot and mouth disease virus monoclonal antibody.
6, a kind of preparation method of aftosa stereotype test strip according to claim 1 is characterized in that being prepared from by following steps:
The preparation of a, collaurum: adopt trisodium citrate reduction method to prepare collaurum;
The O of b, colloid gold label, A, C, Asia I type foot and mouth disease virus Antibody Preparation: at first a is gone on foot the collaurum 0.1mol/L K that makes
2CO
3The adjustment pH value is 8.0-8.5; Determine that with ocular estimate collaurum and antibody to be marked answers the optimised quantity of mark then, obtaining the optimum mark amount is 4-10mg/100mL, presses 4-10mg/100mL and add foot and mouth disease virus antibody in colloidal gold solution, stirs 10～20min; In colloidal gold solution, press 0.5-1g/100ml and add bovine serum albumin(BSA), continue to stir 10～20min, above-mentioned collaurum through the centrifugal 10-20 of 2000-4000r/min minute, is removed sediment, get supernatant; Supernatant is obtained sediment through 10000～12000r/min high speed centrifugation 1h, precipitation is suspended in the gold size damping fluid of the initial collaurum volume of 1/20-1/10,4 ℃ of preservations are put in the dissolving that suspends;
C, the assembling of typing diagnosis test paper:
C.1 preparation contains the nitrocellulose filter that detects band and quality control band: will resist rabbit, cavy and mouse IgG antibody and O, A, C, Asia I foot-and-mouth disease antibody to be adjusted to 1-2mg/ml and 1.4-2.2mg/ml working concentration respectively with pH 7.2-7.6 0.02mol/L phosphate buffer, by 1.8-2ul/cm the spray film is set respectively, above-mentioned two kinds of antibody are sprayed on the nitrocellulose filter, form quality control band and detect band, 37 ℃ of oven dry or after room temperature dries, place confining liquid to soak 5-15min, take out under the room temperature of back and dry, preserve standby;
C.2 prepare the collaurum pad: get all-glass paper, by 10-20 μ L/cm O, A, C, the Asia I foot-and-mouth disease antibody of colloid gold label are sprayed at respectively on the all-glass paper, dry back is stored standby;
C.3 the assembling of test strips: at PVC backer board (1) from the bottom to top respectively with sample pad (5), collaurum pad (4), comprise that the nitrocellulose filter (2) and the absorption pad (3) that detect band (7), quality control band (6) assemble in order, cut into strip, promptly make different shaped foot and mouth disease virus diagnosis test paper, aftosa O, A, C, four kinds of test strips of Asia I type are packed into as a packing and are aftosa typing diagnosis test paper in the aluminium plastic bag.
7, the preparation method of aftosa stereotype test strip according to claim 6 is characterized in that described gold size damping fluid is: the phosphate buffer that contains the PH7.4-7.6 0.11mol/L of 5-15% sucrose, 0.5-1%BSA, 0.5-1 ‰ PEG20000,0.5-1 ‰ Tween-20.
8, the preparation method of aftosa stereotype test strip according to claim 6 is characterized in that described confining liquid is for containing the 2.5-5% calf serum, the phosphate buffer of the PH7.4-7.6 0.02mol/L of 0.5-1 ‰ Tween-20.
9, a kind of method of using aftosa stereotype test strip as claimed in claim 1 is characterized in that using according to the following steps:
The processing of a, sample to be checked: sterile working is washed blister skin to be checked or suckling mouse tissue 2-3 time with the phosphate buffer of PH7.2-7.50.02-0.05mol/L, and inhale the part of anhydrating with sterilized filter paper and weigh, adding glass sand grinds, by 1: 3～1: 10w/v adds the phosphate buffer of PH7.2-7.5 0.02-0.05mol/L, makes suspension; Room temperature is soaked in poison 2 hours or 4 ℃ of refrigerators and is soaked a malicious night. and with the centrifugal 10min of 4000r/min, get supernatant and be sample to be checked after the jolting; Blister liquid to be checked can be directly as sample to be checked;
B, sample detection: sample to be checked, detect test paper and other and detect with material, have an end of markings to insert in the sample to be checked detecting test paper all at equilibrium at room temperature, after liquid all soaks nitrocellulose filter, observations in 5-15 minute;
C, result judge
C.1 single test strips result judges:
Positive: quality control band (6) high-visible red stripes all occurs with detection band (7);
Negative: as to have only quality control band (6) high-visible red stripes to occur;
Suspicious: the apparent red stripes of color appears in quality control band (6), and is detecting light color band of band (7) appearance;
Invalid: red stripes does not appear in quality control band (6);
C.2 the result that finalizes the design judges:
The O type: the result is positive for O type test strips, and the result is negative or suspicious for Asia I, A, C type test strips;
Asia I type: the result is positive for Asia I type test strips, and the result is negative or suspicious for O, A, C type test strips.
The A type: the result is positive for A type test strips, and the result is negative or suspicious for O, Asia I, C type test strips.
The C type: the result is positive for C type test strips, and the result is negative or suspicious for O, Asia I, A type test strips.
Negative: the result is all negative for O, Asia I, A, C type test strips.
Suspicious: O, Asia I, A, C type test strips result are suspicious, or wherein three kinds, two kinds test strips results are suspicious.
When four kinds, three kinds and two kinds of test strips are all positive to same antigen colour developing result to be checked, sample to be checked can be the two-fold dilution with dilution, detect again, judge c.2 to carry out the result.
10, the using method of aftosa stereotype test strip according to claim 9 is characterized in that described dilution is the PH7.4-7. 60.11Mol/L phosphate buffer that contains 0.5-1 ‰ Tween-20.
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