CN103940994A - Swine vesicular disease, swine vesicular exanthema and swine vesicular stomatitis tri-combination detection test paper strip - Google Patents
Swine vesicular disease, swine vesicular exanthema and swine vesicular stomatitis tri-combination detection test paper strip Download PDFInfo
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Abstract
本发明涉及一种猪三种疫病的检测试剂显示的器具，特别是涉及一种猪水疱病、猪水疱性疹及猪水疱性口炎快速诊断试纸条，试纸条含支撑层、反应试剂载体吸附层，支撑层为不吸水薄片条，反应试剂载体吸附层粘贴于支撑层上，从样品测试端依次为纤维层，上述三种疫病抗原金标单抗或多抗纤维层，纤维素膜层，手柄端为吸水材料层；分别用上述三种疫病抗原配对单抗或多抗或单抗溶液在纤维层上喷检测印迹“｜｜｜”、“///”、或“＼＼＼”，分别用羊（兔）抗小鼠或猪IgG的多抗或用SPA溶液在纤维素膜层上喷对照印迹“｜”、“/”或“＼”。该试纸条特异、敏感、直观、准确、简便、快速，能在畜禽饲养、肉类加工和检疫等相关部门推广应用。
The invention relates to a device for displaying detection reagents of three kinds of pig diseases, in particular to a rapid diagnosis test strip for porcine vesicular disease, porcine vesicular rash and porcine vesicular stomatitis. The test strip contains a support layer and a reaction reagent carrier for adsorption The support layer is a non-absorbent sheet strip, and the reaction reagent carrier adsorption layer is pasted on the support layer. From the sample test end, there is a fiber layer, the above three epidemic antigen gold standard monoclonal antibody or polyantibody fiber layer, cellulose film layer, The end of the handle is a water-absorbing material layer; use the above-mentioned three kinds of epidemic antigen paired monoclonal antibody or polyclonal antibody or monoclonal antibody solution to spray the detection imprint "｜｜｜", "///", or "＼＼＼" on the fiber layer, Use goat (rabbit) anti-mouse or pig IgG polyclonal antibody or SPA solution to spray control blots "|", "/" or "\" on the cellulose membrane layer respectively. The test strip is specific, sensitive, intuitive, accurate, simple and fast, and can be popularized and applied in relevant departments such as livestock and poultry breeding, meat processing and quarantine.
Description
one, technical field
The present invention is that one relates to SVD, pig blister rash and pig vesicular stomatitis detection reagent displaying appliance, particularly relates to a kind of three test strip that can simultaneously detect SVD, pig blister rash and pig vesicular stomatitis.
two, technical background
SVD (Swinevesiculardisease, SVD) claims again swine vesicular disease, is to cause the acute contagious disease of one of pig by swine vesicular disease virus (Swine vesicular diseasevirus, SVDV).The popularity of this disease is strong, the incidence of disease is high, occur that mainly with coronet, frog blister is feature clinically, also there is blister at lip, tongue, oral area, nose and belly and nipple surrounding skin once in a while, often fall ill with popular form, harm to pig industry is larger, thereby it prevents and treats the problem utmost point and paid attention to, and OIE (OIE) is classified as animal category-A infectious disease.Swine vesicular disease virus (Swine vesicular disease virus, SVD) be a member of Picornaviridae enterovirus genus, with human coxsackievirus Coxsaekievirus) especially Coxsackie B virus 5 (CoxsackievirusB5, CVB5) have a lot of similaritys at aspects such as physicochemical property, biological characteristics and serological relations.SVD finds in Italy for 1966 first, and nineteen sixty-eight separates and confirmed SVDV.Clinical symptoms is very similar to aftosa, pig blister rash and pig vesicular stomatitis, but the artiodactyl such as infected cattle, sheep not.Sick pig and recover after be the main infection sources of this disease with malicious pig, mainly through wound and infection of digestive canal, virus is got rid of by excrement, urine, blister liquid and blister skin.The pig of different cultivars, age, sex all can occur, but the most responsive with sucking pig and young pig.When natural infection, be 2 ~ 4 days latent period, body temperature reaction that their early stage is slight.At the bottom of coronet, frog, hoof, there is blister: pale swelling of initial stage, within 36～48 hours, blister is protruded, and has the grain of rice large to soya bean, is full of limpid or weak yellow liquid in blister, and finger pressure is slightly soft; Through 1～2 day ulceration, form ulcer surface.Severe one coffin and coronet split, come off.If secondary bacterium infects, hoof is undermined.Disease pig pain, fortune walk difficulty, walk lamely or creep, or crouch and do not rise, poor appetite or useless exhausted.The sick pig of part has blister on nose dish, but occurs more late.Sometimes also there is blister and ulcer at lip, lingual surface, palate and nipple.After newborn piglet morbidity, can cause death.Cut open the distinctive pathology of inspection at hoof, internal organs are without naked eyes pathology.
Pig blister rash is acute, the hot infectious disease of one of the pig that caused by pig blister exanthema virus.It is characterized in that oral mucosa and hoof skin generation blister, after ulceration, become ulcer.Virus is single-stranded RNA type virus, and diameter is 30～40nrn.Virus can be at pig kidney can separate cultivation on primary or passage cell, just produces cytopathy through 48～72 hours.This viral resistance is stronger,, can survive 6 weeks in room temperature through the deactivation of 30 points of ability in 60 DEG C.More responsive to alkaline sanitizer.Sick pig is the main infection sources, infects this disease by introducing infected pigs and products thereof.The propagation of this disease is mainly directly to be contacted and cause with strong pig by sick pig, or the contaminated feed of feeding, meat product rubbish, butchers leftover bits and pieces, and causes propagation without the approach such as kitchen odds and ends of a meal, swill boiling.Natural infection only betides pig, all ages and classes, sex and kind no significant difference.Can there is blister in hamster and dog intradermal vaccination.The generation of this disease is without obvious seasonality.What the incidence of disease was low only has 10%, and high reaches 100%, and its difference is relevant with neurological susceptibility, feeding and management and the virus type etc. of pig.Be 1～7d latent period.Sick just body temperature rises to 40.5～41.5 DEG C of lasting a few days.Spirit is depressed, poor appetite.Subsequently between nose dish, lip, oral cavity, sole of the foot portion, toe, there is successively canescence blister and cherry ulcer surface in coronet, the first-class position of sow milk.Severe patient can not be walked, and pain is yelped, and even coffin comes off, and salivation, food refusal.As without scabies secondary infection, how rehabilitation in 1 week.What pregnant pig had miscarries.Can there is death in minority suckling pig.General prognosis bona, case fatality rate 5% left and right.Part pig is subclinical infection and resistance to mistake, but band is malicious over a period to come.Suffer from this pig after being ill homologous virus is had to immunity.
Vesicular stomatitis is the acute hot infectious diseases common to human beings and animals of one being caused by vesicular stomatitis virus (Vesicular stomatitis virus, VSV).This virus belongs to Rhabdoviridae (Rhabdovifidae) vesiculovirus genus (Vesiculovirus), is the sub-thread minus-stranded rna virus that has cyst membrane.VSV is the Important Infectious Diseases that causes the many animals such as ox, horse, pig and people's coinfection, and the cause of disease of vesicular stomatitis is all significant animal doctor and field of public health.VSV is and has a liking for epithelium, and infected domestic animal main manifestations is mouthful, hoof and the infringement of peripapillary blister sample, and meat yield and the output of milk decline, and generally do not cause animal dead.People infects the symptom of the similar influenza of rear appearance, but can not cause blister.When VSV infected cattle and pig, clinical symptoms and aftosa, SVD, pig vesicle are difficult for difference, and this disease makes the productive capacity of pig decline and cause serious economic loss, and international trade is produced and had a strong impact on, have important social economy and public health meaning.OIE (OIE) classifies vesicular stomatitis (Vesicular stomatitis, VS) as legal circular infectious disease.This virus all can be bred on 7～13 age in days chick chorioallantoic membranes He in allantoic cavity, and lethal chicken embryo in the time of 24～48 hours.Can also in the kidney of many animals or epithelium primary cell and the cultivation of multiple passage cell, breed well, and produce cytopathy.Route of transmission is mainly skin and the mucous membrane of damage, in swinery, mainly propagates with the way of contact, instead of through infection of digestive canal.Dipteral insect is the important communication media of this disease, and sand fly, yellow-fever mosquito and mite are all separated to this virus.Can make the various animal morbidities including animal used as test by artificial infection.When natural infection, be 2～5 days latent period, and artificial challenge mostly is 1 day.40～41 DEG C of sick just hyperthermias, spirit is depressed, anorexia and salivation.Sick hog snout portion blister is comparatively common, and hoof blister is mainly in frog and rare in coronet portion, includes yellow transparent liquid.Blister appears ulcer surface after breaking, sick pig dysstasia, and occur walking lamely.
These three kinds sick clinical symptoms clinical symptoms are similar, and defficulty in diagnosing has brought difficulty to the control of this type of disease.Differentiating that fast detection can be the morbidity treatment of swinery and the removing of pathogen provides scientific basis, is the effective ways of controlling and eliminate above-mentioned disease.At present the diagnostic method of above-mentioned disease is mainly contained following several.
(1) separation of virus is cultivated: get after typical blister tissue and liquid homogenate dilution thereof, add 4 DEG C of effects of antibiotic 4 hours, 0.22mm membrane filtration, filtrate being inoculated in is paved with on the porcine kidney cell or porcine kidney cell primary cell of individual layer, 37 DEG C adsorb 1 hour, add maintenance medium and continue to cultivate 48 ~ 72 hours observation of cell pathologies.
(2) animal inoculation pvaccination test: get the rear 0.22mm membrane filtration of pathological material of disease homogenate dilution, filtrate is inoculated in respectively different days suckling mouse.Continuous Observation records the death condition of suckling mouse.Take a decision as to whether SVD, vesicular stomatitis, pig blister rash according to the death condition of suckling mouse.
(3) serology detects: reverse indirect hemagglutination assay, availablely defend the fixing sheep red blood cell (SRBC) of dialdehyde one formaldehyde and with the anti-hydroa antibody sensitized of cavy, make sensitized erythrocyte diagnosticum, diagnosticum and antigen to be checked (blister liquid or blister skin) carry out reverse indirect hemagglutination assay, within 2～7 hours, can obtain a result.Neutralization test, get 8 of 1～2 age in days suckling mouses, each 4 of test group and control group, (pathological material of disease grinds and makes 1:5 dilution with physiological saline for anti-hydroa serum and tested material for test group, every milliliter adds dual anti-, put 4 DEG C of refrigerators and soak poison 6 hours, get supernatant) be above-mentioned leachate mixed in equal amounts, control group physiologic saline for substitute antiserum, put in 37 DEG C of incubators and act on 1 hour, every suckling mouse neck hypodermic injection 0.l milliliter, as all dead in control group suckling mouse, and the survival of test group suckling mouse proves in this sample containing standby inspection virus; Or get standard positive serum and aseptic blister liquid, and adopt Small Volume Serum neutralisation, on BHK-21 passage cell, carry out neutralization test, can be neutralized by standard positive serum, there is not pathology person in cell, is judged to the positive.Agar diffusion test, this law is for checking the specific antibody of the doubtful pig serum of hydroa.All can reacting with standard antigen, producing obvious sediment line person can make a definite diagnosis.
(4) inverse transcription polymerase chain reaction (RT-PCR) technology: RT-PCR technology has the advantage such as high sensitivity, high specific, has been widely used Causal Agent Identification and epidemiology survey in numerous disease.The pathogen detection of above-mentioned disease all can be used RT-PCR technology to detect.
Separation cultivation, the serology of virus detects, inverse transcription polymerase chain reaction (RT-PCR) technology, needs professional in laboratory operation, complex operation, and detection is wasted time and energy; And need expensive instrument and equipment, as PCR instrument, microplate reader and CO
2incubators etc., for layman, above-mentioned detection method has been difficult to.Although the special sensitivity of said method, can only detect a kind of swine disease, and cannot realize field quick detection or diagnosis at every turn.The present invention, research a kind of easy fast, real-time online, detect three joint inspection test papers of three kinds of swine diseases, to controlling and to eliminate this type of disease significant simultaneously.
three, summary of the invention
The object of the invention is the shortcoming that detects the existence of swine disease cause of disease in prior art in order to overcome, a kind of special, responsive, simple and rapid breathing problem detection method is provided, develops the three joint inspection test papers that once can simultaneously detect three kinds of pig bacterial respiratory tract diseases.
Technical scheme of the present invention is: a kind of SVD, pig blister rash and pig vesicular stomatitis three test strip are provided, this test strips contains supporting layer and adsorbed layer, supporting layer is the lamella not absorbing water, adsorbed layer is attached on supporting layer, adsorbed layer is followed successively by the absorbent material layer of sample adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete and handle end from test lead, be provided with and detect trace and contrast trace on cellulose rete; The absorption of gold labeling antibody fibrage has three kinds of monoclonal antibodies of anti-SVD, pig blister rash and the pig vesicular stomatitis pathogen specific antigen of nanometer grade gold particle marker, detect the pairing monoclonal antibody of anti-SVD, pig blister rash and pig vesicular stomatitis pathogen specific antigen for trace and print, the polyclonal antibody of goat-anti or the anti-mouse IgG of rabbit for contrast trace; Or golden labeling antibody fibrage absorption has the polyclonal antibody of anti-SVD, pig blister rash and the pig vesicular stomatitis pathogen of nanometer grade gold particle marker, detect trace respectively with the monoclonal antibody preparation of anti-SVD, pig blister rash and pig vesicular stomatitis pathogen specific antigen, contrast staphylococcal protein A (SPA) or the how anti-preparation of anti-pig IgG for trace.
Detecting the pairing monoclonal antibody preparation of anti-SVD, pig blister rash and pig vesicular stomatitis pathogen specific antigen for trace prepares respectively with the pairing monoclonal antibody solution of above-mentioned three kinds of pathogen specific antigens; The polyclonal antibody that detects anti-SVD, pig blister rash and pig vesicular stomatitis pathogen trace for is prepared to be and is prepared respectively with the polyclonal antibody of above-mentioned three kinds of pathogen.
Supporting layer is made with the hard plastic slip or the cardboard bar that do not absorb water; Test lead sample adsorbing fiber layer is made with glass wool; Gold labeling antibody fibrage is made with glass wool and golden labeling antibody, and golden labeling antibody can be monoclonal antibody or polyclonal antibody.
Cellulose rete is made with nitrocellulose filter or pure cellulose film or carboxylation cellulose membrane or polyvinylidene fluoride PVDF cellulose membrane.
Absorbent material layer is made with thieving paper.
Detect trace and contrast trace is orthoscopic or oblique line formula, on cellulose rete, contain three and detect traces and one contrast trace, the spread pattern that detects trace and contrast trace be "
|| ||", "
// //", "
?" in any.
Above test strips adsorbed layer, contain layer protective layer; protective seam is attached on adsorbed layer; on test lead sample adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer, be coated with diaphragm; on the test lead sample adsorbing fiber layer diaphragm corresponding with golden labeling antibody fibrage intersection, be printed with sample mark line, this mark line deflection test lead sample adsorbing fiber layer one about 0.5cm place of side place.
As required, select above-mentioned golden labeling antibody fibrage, detect a kind of form in trace and contrast trace spread pattern.
Positive beneficial effect of the present invention:
1. detection specificity is strong, susceptibility is high: test strip of the present invention is made as basis taking nanometer grade gold particle marker high-affinity monoclonal antibody specific or specific polyclonal antibody, in gold labeling antibody, between gold grain and antibody molecule, form without covalent bond, the two combines by the Van der Waals force between the charges of different polarity, gold grain does not affect specificity and the adhesion of monoclonal antibody or polyclonal antibody, and has higher mark rate.Test strip of the present invention has higher specificity and susceptibility, nanogram level pathogen albumen can be detected.
2. easy and simple to handle, quick: to use when ELISA test strip of the present invention without additional any Other Instruments and reagent, its test lead need be inserted in sample liquid to be checked about 30 seconds, then in 1-5 minute, can judge testing result.
3. testing result is directly perceived, accurate: whether test strips of the present invention is to show that henna detection line and control line are as the foundation of judging positive and negative findings, only show a brownish red control line C at the control line marking place of cellulose membrane, and show without brownish red band at detection line marking place, represent that 3 kinds of detected swine diseases are negative findings; Control line marking place at cellulose membrane shows a brownish red control line C, be SVD detecting trace place appearance three brownish red band T1, T2, T3(T1, T2 is pig blister rash, and T3 is pig vesicular stomatitis), represent that 3 kinds of detected swine diseases are positive findings; On cellulose membrane, show arbitrary or wantonly two in a brownish red control line and three brownish red detection lines, be illustrated in any one or two kinds of positive result in detected 3 kinds of swine diseases.No matter positive findings or negative findings control line C all should show, in the time that control line C does not show, illustrate that test strips lost efficacy.
4. testing cost reduces: use test strip of the present invention, do not need Other Instruments and reagent, saved instrument, equipment and additive reagent expense; Article one, test paper once can detect 3 kinds of swine diseases, and layman is real-time online detection at any time also, without paying expert diagnosis Laboratory Fee and correlative charges thereof, can reduce greatly the input of testing cost, reduces testing cost.
5. usable range is wide: test strip of the present invention is simple to operate, i.e. " foolproof " operation, and easy to carry, easily preservation, can meet the not needs of commensurate and different levels personnel, comprise that professional chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, livestock products processing, intensive culture arrive individual cultivation etc., have wide market outlook and social benefit.
four, brief description of the drawings:
The side-looking structural representation of a kind of SVD of Fig. 1, pig blister rash and pig vesicular stomatitis three test strip
The plan structure schematic diagram of a kind of SVD of Fig. 2, pig blister rash and pig vesicular stomatitis three test strip
five, embodiment:
Following examples only, in order to further illustrate the present invention, do not limit content of the present invention.The preparation of SVD, pig blister rash and pig vesicular stomatitis three test strip, needs to prepare the monoclonal antibody and the polyclonal antibody that resist three kinds of pathogen specific antigens, for the preparation of detecting trace and golden labeling antibody fibrage; Need to prepare sheep or rabbit anti-mouse igg antibody simultaneously, or sheep or the anti-pig IgG antibody of rabbit, for the preparation of contrast trace.
1. the preparation of the anti-mouse of sheep (rabbit) or pig IgG antibody:
Extract the IgG in mouse or pig serum with saturated ammonium sulfate method, get 1 part of serum and add 2 parts of PBS liquid (pH 7.2) and mix, add equal-volume saturated ammonium sulfate liquid and mix, put 2h in 4 DEG C of refrigerators, at 4 DEG C, the centrifugal 15min of 10000r/min, abandon supernatant; With appropriate PBS liquid (pH7.2) dissolution precipitation, adding saturated ammonium sulfate liquid to its ultimate density is 33%, put 2h in 4 DEG C of refrigerators, centrifugal 15min under 4 DEG C, 10000r/min condition, abandons supernatant, with a small amount of PBS liquid (pH7.2) dissolution precipitation, put in 4 DEG C of refrigerators with PBS liquid (pH7.2) dialyzed overnight, change liquid 2～3 times, centrifugal 15min under 4 DEG C, 10000r/min condition, collect supernatant, measure its protein concentration with ultraviolet spectrophotometer.With 50 μ g～100 μ g(IgG)/kg body weight is through subcutaneous or intramuscular injection negative antibody Healthy Sheep or rabbit 3～4 times, last immunity is after 20 days, venous blood collection, measure its serum antibody titer more than 1:2000 with ELISA, heart blood sampling or arteria carotis bloodletting, collect its hyper-immune serum, and its extracting method of IgG(that extracts the anti-mouse of sheep (rabbit) or pig with saturated ammonium sulfate method is identical with said extracted mice serum IgG, no longer repeat), for the preparation of the contrast trace of test strips of the present invention.
2. the preparation of SVD, pig blister rash and pig vesicular stomatitis pathogen specific antigen monoclonal antibody (Mi):
Every use 50 μ g～100 μ g pathogen specific antigen immunity Balb/c are mouse three times, every minor tick 15～30d; 3～4d after booster immunization for the third time, by the bloodletting of immune mouse eyeball, draws neck lethal, soaks blister 5～10min with 75% alcohol, and aseptic its spleen of getting, shreds and through 100 order nylon net filters, the centrifugal 10min of 1000r/min, collects splenocyte; By 1 × 10
8individual splenocyte and 2～5 × 10
7individual NS0 myeloma cell mixes, 1000r/min is centrifugal, and 10min abandons supernatant, the centrifuge tube that contains sedimentation cell is placed in to the water of 37 DEG C, and slowly add 0.7～1ml, 40%～50% PEG4000(pH 8.5～9.0) effect 1min, then slowly add serum-free 1640 nutrient culture media 15ml, to stop the effect of PEG, 37 DEG C of water-bath 5～10min, 1000r/min is centrifugal, and 10min abandons supernatant, cell precipitation is resuspended in to HAT and selects in nutrient culture media, and add
96 well culture plates (100 μ l/ hole, μ l～200), are placed in 37 DEG C of 5% CO
2in incubator, cultivate.Cultivate after 7～10d, with the coated 96 hole ELISA Plate of pathogen specific antigen of the purifying of 5 μ g～10 μ g/ml, detect the culture supernatant of hybridoma with enzyme linked immunosorbent assay (ELISA), picking strong positive cell clone (OD
450>=0.5), carry out the limiting dilution assay cloning of continuous three times, obtain positive hybridoma cell strain, its chromosome number is 92～98, the monoclonal antibody (M1, M2 or M3) of its secretion, can three kinds of different pathogen of specific recognition, and not with the pathogen generation cross reaction of other pig, affinity constant reaches 10
9 ~ 10, light chain subtype is к or λ, heavy chain hypotype is IgG
1, IgG
2a, IgG
2b, IgG
3; The pairing monoclonal antibody obtaining, for making gold mark monoclonal antibody body glass wool or detecting trace.
3. the preparation of gold mark monoclonal antibody glass wool:
Utilize sodium citrate reduction method for preparing nanometer level gold grain: in 0.01～0.05% aqueous solution of chloraurate of 50～100ml boiling, add 0.5～2% citric acid three sodium solution of 2～4ml, obtain the nanometer grade gold particle of diameter 15nm left and right.With the K of 0.1mol/L
2cO
3adjust pH to 8.5～9.5 of gold grain solution, mark with 1:1000～1300 compares monoclonal antibody to be marked (M1, M2 or M3) add in the aurosol of pH8.5～9.5, after mark 10min, add 20% PEG10000 to ultimate density be 0.05%, 4 DEG C, the centrifugal 20min of 1500～3000r/min, remove unconjugated gold grain particle, 4 DEG C, the centrifugal 1h of 15000r/min, abandon supernatant, obtain after golden labeling antibody potpourri, with propylene glucosan S-400 column chromatography, separation and purification gold labeling antibody, obtains respectively the golden labeling antibody of M1, M2, M3.By three kinds of golden labeling antibodies of 1:100～500 dilution, be adsorbed in processed glass cotton 4 DEG C of low-temperature vacuum dryings, preparation gold mark monoclonal antibody glass wool.
4. the preparation of pathogen specific antigen polyclonal antibody (Ci):
The preparation of pathogen specific antigen polyclonal antibody (Ci).Adopt respectively inactivated vaccine, attenuated vaccine or the standard antigen of above-mentioned three kinds of swine diseases of state approval, repeatedly immunity inoculation negative antibody health pig.Last immunity posterior vein blood sampling in 20 days, measure its serum antibody titer more than 1:2000 with ELISA, heart blood sampling or arteria carotis bloodletting, collect its hyper-immune serum, extract IgG antibody (method is identical with the extraction of mice serum IgG, no longer repeats) in serum with saturated ammonium sulfate method.
Gold mark resists the preparation with the how anti-glass wool of gold mark more, identical with the preparation method of gold mark monoclonal antibody glass wool, no longer repeats.
Refer to the content 3 in embodiment.
5. test strip of the present invention detects principle
When test strip test lead of the present invention inserts after sample solution to be checked, solution to be checked drives pathogen to be checked to enter golden labeling antibody fibrage by siphon, and spread to handle end along nitrocellulose filter with together with golden labeling antibody (Mi or Ci) wherein, the final handle end absorbent material layer that infiltrates, in diffusion process, golden labeling antibody can be combined with corresponding pathogen to be checked, in conjunction with the pathogen of golden labeling antibody can on cellulose membrane, be detected pairing monoclonal antibody or the how anti-interception of trace, in the time containing tested pathogen in sample liquid, there is 1～3 henna detection line, the anti-mouse of sheep or rabbit or anti-pig IgG can be marked monoclonal antibody or many anti-bindings with corresponding gold, occur 1 brownish red control line.In the time there is no above-mentioned pathogen in sample liquid to be checked, test strips only demonstrates a brownish red control line; In the time not having control line to show on cellulose membrane, show that test strips lost efficacy.
6. the detection method of operating of test strip of the present invention
(1) detect the processing of sample: get disease pig pathological tissues, 1:1～5 add physiological saline and shred with scissors, and leachate is sample to be checked, and sick pig whole blood or serum are sample to be checked after adding the dilution of physiological saline 1:1～5.
(2) detect operation: test strip sample end of the present invention is inserted in sample liquid to be checked, and insertion depth is no more than mark line 9, after approximately 30 seconds, takes out test strips, horizontal positioned approximately 1～5 minute, simultaneously observations.
(3) result is judged: if only demonstrate a brownish red control line C on test strip cellulose membrane, represent that testing result is negative, illustrate in test sample not containing above-mentioned 3 kinds of pathogen; If there is control line C in the cellulose membrane in test strip, detect trace place and occur T1 or T2 or T3 detection line, represent that testing result is positive, in sample to be checked, contain SVD pathogen or pig blister exanthema substance or pig vesicular stomatitis pathogen; Occur if detect the T1 of trace place or T2 or T3 simultaneously, be illustrated in and in sample to be checked, have above-mentioned 3 kinds of pathogen; If show without any brownish red trace on cellulose membrane, show that test strips lost efficacy.
Embodiment mono-: SVD, pig blister rash and pig vesicular stomatitis three test strip
Referring to Fig. 1 and Fig. 2, in figure, 1 is supporting layer, make by hard plastic strip of foil, 2 is the sample adsorbing fiber layer of test lead, make with glass wool, 3 is golden labeling antibody fibrage, absorption has the anti-SVD of nanometer grade gold particle marker, the glass wool of three kinds of monoclonal antibodies of pig blister rash and pig vesicular stomatitis pathogen, prepare its gold mark monoclonal antibody glass wool according to the preparation method described in above-mentioned embodiment 3, 4 is cellulose rete, employing nitrocellulose filter is made, 5 is absorbent material layer, make with thieving paper, to number 2, 3, 4, 5 each layers stick on hard plastic strip of foil 1 from left end test lead to the right side, intersection crosses one another overlapping each other.On cellulose nitrate rete 4,6 is detection trace T1, T2, the T3 that the pairing monoclonal antibody solution by anti-SVD, pig blister rash and pig vesicular stomatitis pathogen is printed respectively, 7 is the contrast trace C printing with sheep or rabbit anti-mouse igg solution, detect trace and contrast trace is orthoscopic or oblique line formula, two kinds of trace bands arrange the array configuration forming be "
|| ||", "
// //", "
?" in any.8-1 covers test lead sample adsorbing fiber layer 2 and golden labeling antibody fibrage 3 white diaphragm above; on the corresponding diaphragm 8-1 of 2 and 3 intersections position, be partial to sample adsorbing fiber layer 2 one side 0.5cm place and be printed on mark line 9; 9 right-hand member is printed on arrow and max printed words, absorbent material layer 5(handle end) on be coated with other color (as yellow) diaphragm 8-2.
The preparation of testing sample solution and detection operation steps, identical with the detection method of operating in embodiment 6, no longer repeat.
Embodiment bis-: SVD, pig blister rash and pig vesicular stomatitis three test strip, basic identical with embodiment mono-, difference is:
The 3 use absorption of gold labeling antibody fibrage have the glass wool of three kinds of polyclonal antibodies of anti-SVD, pig blister rash and the pig vesicular stomatitis of gold grain mark to make, and prepare its gold mark polyclonal antibody glass wool according to the preparation method described in above-mentioned embodiment 3; On cellulose nitrate rete 4,6 is detection trace T1, T2, the T3 that the monoclonal antibody solution by anti-SVD, pig blister rash and pig vesicular stomatitis pathogen is printed respectively, 7 is to print contrast trace C with sheep or the anti-pig IgG solution of rabbit, two kinds of trace bands arrange the array configuration forming be "
|| ||", "
// //", "
?" in any.Other comprises that detection sample preparation, method of operating and result judgement etc. are all identical with the method for operating in embodiment 6, no longer repeats.
Claims (8)
1. one kind is detected SVD, three test strip of pig blister rash and pig vesicular stomatitis, this test strips contains supporting layer and adsorbed layer, supporting layer is the lamella not absorbing water, adsorbed layer is attached on supporting layer, adsorbed layer is followed successively by sample fiber layer from test lead, gold labeling antibody fibrage, the absorbent material layer of cellulose rete and handle end, on cellulose rete, be prepared with and detect trace and contrast trace, it is characterized in that golden labeling antibody fibrage absorption has the anti-SVD of nanometer grade gold particle marker, three kinds of monoclonal antibodies of pig blister rash and pig vesicular stomatitis pathogen specific antigen, detect the anti-SVD of trace, the pairing monoclonal antibody of pig blister rash and pig vesicular stomatitis pathogen specific antigen or polyclonal antibody are printed, contrast trace is printed with polyclonal antibody or the staphylococcal protein A (SPA) of sheep or the anti-mouse IgG of rabbit.
2. test strips according to claim 1, it is characterized in that golden labeling antibody fibrage absorption has the anti-SVD of nanometer grade gold particle marker, the mixed liquor of three kinds of monoclonal antibodies of pig blister rash and pig vesicular stomatitis pathogen specific antigen, detect the anti-SVD of trace, the pairing monoclonal antibody of pig blister rash and pig vesicular stomatitis pathogen specific antigen or polyclonal antibody are printed, contrast trace is printed with polyclonal antibody or the staphylococcal protein A (SPA) of sheep or the anti-mouse IgG of rabbit, or golden labeling antibody fibrage absorption has the anti-SVD of nanometer grade gold particle marker, the polyclonal antibody of pig blister rash and pig vesicular stomatitis pathogen, detect trace and use respectively anti-SVD, the monoclonal antibody preparation of pig blister rash and pig vesicular stomatitis pathogen specific antigen, contrast staphylococcal protein A (SPA) or how anti-preparation of anti-pig IgG for trace.
3. test strips according to claim 1 and 2, the pairing monoclonal antibody preparation that it is characterized in that detecting anti-SVD for trace, pig blister rash and pig vesicular stomatitis pathogen specific antigen is prepared respectively with the pairing monoclonal antibody solution of above-mentioned three kinds of pathogen specific antigens; The polyclonal antibody that detects anti-SVD, pig blister rash and pig vesicular stomatitis pathogen trace for is prepared to be and is prepared respectively with the polyclonal antibody of above-mentioned three kinds of pathogen.
4. test strips according to claim 1, is characterized in that supporting layer the hard plastic slip or the cardboard bar that do not absorb water make; Test lead sample adsorbing fiber layer is made with glass wool; Gold labeling antibody fibrage is made with glass wool and golden labeling antibody, and golden labeling antibody can be monoclonal antibody or polyclonal antibody.
5. test strips according to claim 1, is characterized in that cellulose rete nitrocellulose filter or pure cellulose film or carboxylation cellulose membrane or polyvinylidene fluoride PVDF cellulose membrane make.
6. test strips according to claim 1, is characterized in that absorbent material layer thieving paper makes.
7. test strips according to claim 1, is characterized in that detecting trace and contrast trace is orthoscopic or oblique line formula, on cellulose rete, contain three and detect traces and one contrast trace, the spread pattern that detects trace and contrast trace be "
|| ||", "
// //", "
?" in any.
8. test strips according to claim 1; it is characterized in that containing layer protective layer above test strips adsorbed layer; protective seam is attached on adsorbed layer; on test lead sample adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer, be coated with diaphragm; on the test lead sample adsorbing fiber layer diaphragm corresponding with golden labeling antibody fibrage intersection, be printed with sample mark line, this mark line deflection test lead sample adsorbing fiber layer about 0.5cm of one side place.
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