CN106198970A - A kind of test kit of quick detection H9N2 subtype avian influenza virus - Google Patents
A kind of test kit of quick detection H9N2 subtype avian influenza virus Download PDFInfo
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- CN106198970A CN106198970A CN201610528528.5A CN201610528528A CN106198970A CN 106198970 A CN106198970 A CN 106198970A CN 201610528528 A CN201610528528 A CN 201610528528A CN 106198970 A CN106198970 A CN 106198970A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention discloses the test kit of a kind of quick detection H9N2 subtype avian influenza virus, including box body and detection card, described box body includes sample collecting district and results display area, described detection card includes base plate, absorbent filter, colour developing pad and sample pad, described colour developing pad includes detection zone and quality control region, described sample pad includes neonychium and colloid layer gold, described colloid layer gold is made up of the glass fibre membrane of the monoclonal antibody of the H9N2 subtype avian influenza virus scribbling colloid gold particle labelling, described detection zone is coated with the polyclonal antibody of H9N2 subtype avian influenza virus, described quality control region is coated with rabbit anti-mouse igg antibody.The test kit of the present invention, simple to operate, specificity is good, can obtain testing result in 5 10min, it is simple to whether birds is infected H9N2 subtype avian influenza virus and makes quick diagnosis, have good dissemination.
Description
Technical field
The present invention relates to animal infectious disease detection apparatus technical field, be specifically related to a kind of quickly detection H9N2 sub-
The test kit of type bird flu virus.
Background technology
According to the relevant laboratory statistical data of country, what commercial meat bird sense epidemic disease dye rate in 2012 was the highest is still that H9N2.
If added up by H9N2 with H5, total infection rate is 82.21%.The reason of this every chicken death of explanation, has 82.21% source
In bird flu.And only accounted for 17.79% by what other diseases caused.In commercial meat bird, hybrid meat chicken, young chicken group, according to country
Shutting mechanism data is had to prove, H9N2 influenza virus (AIV) latent infection rate 1%.Do not fix after individually infecting H9N2
Clinical manifestation symptom.Tested by virus inoculation, can't see any pathological changes and the symptom of H9N2, once taked the various ways such as intramuscular injection
After counteracting toxic substances, do not find that H9N2 has any clinical manifestation symptom.This explanation H9N2 the most individually infects, the most not pathogenic, only continues
Send out or just can show some clinical symptoms after other disease concurrent.Therefore, should be by when prevention and control H9N2 type bird flu
The disease that treatment is done mischief with it synchronizes to carry out.
The H9N2 influenza morbidity form the most common when winter-spring season is fallen ill is influenza secondary airsacculitis form, in pathological changes
Main performance part chicken swell head swell face, draw green feces, have respiratory symptom, eyes to shed tears, drop-head eye closing etc..The main table of pathological changes
Now pancreas edge is hemorrhage, thymus swelling is hemorrhage, there are hemal ring, fibrinous pneumonia etc. in tracheorrhagia, trachea bottom.If continued
Sending out after infecting, the serous effusions such as foam occur in mainly showing abdominal cavity, anterior thoracic air-sac, after 2 days, lungs there will be typically
Fibrinous pneumonia.Edema stage of congestion (edema stage of congestion), rubescent hemorrhage (red liver becomes the phase), lungs light gray swelling occur
(Lycoperdon polymorphum Vitt liver become phase), the most hemorrhage fibrinoid necrosis (cellulose sample liver becomes the phase), the disease such as the hemorrhage tuberculous pneumonia in some areas
Become.At each air bag of middle and late stage, intraperitoneal, serious cheesy exudate all can occur simultaneously；Part has cellulose sample to ooze out
Thing.Just constitute so-called " airsacculitis is sick " or " influenza airsacculitis is sick ".
First prevention for H9N2 subtype avian influenza virus is the resistance strengthening chicken at present, can select enhancing immunity
Chinese medicinal formulae keep healthy.General immunity is all to be selected in carry out in first three week, the guarantor of this three weeks 1-2 courses for the treatment of to be carried out
Strong, if do not kept healthy, chicken the most very likely falls ill, secondly for different chicken group's reasonable application of antibiotics；Additionally, exempting from
Must again build up resistance during epidemic disease, the detection to H9N2 subtype avian influenza virus simultaneously is also most important, especially for basic unit
Animal epidemic disease control worker for, quickly detect virus have the most crucial effect for preventing from timely infecting.
Summary of the invention
For the problems referred to above, the invention provides a kind of quickly detection H9N2 hypotype fowl stream highly sensitive, easy to use
The test kit of Influenza Virus.
The technical scheme is that the test kit of a kind of quick detection H9N2 subtype avian influenza virus, including box body and
Detection card, described box body includes sample collecting district and results display area；Described detection card was embedded in box body, the end of including
Plate, absorbent filter, colour developing pad and sample pad, absorbent filter, colour developing pad and sample pad are bonded on base plate successively；Described sample
Pad includes neonychium and colloid layer gold, and neonychium is stuck in above colloid layer gold, and described colloid layer gold is by scribbling gold colloidal
The glass fibre membrane of the monoclonal antibody of the H9N2 subtype avian influenza virus of grain labelling is made, and described colour developing pad includes detection zone
And quality control region, described detection zone is coated with the polyclonal antibody of anti-H9N2 subtype avian influenza virus, and described quality control region is coated with
Rabbit anti-mouse igg antibody.
Further, described base plate is that synthetic resin material prepares, and described synthetic resin is macromolecular compound, is
By low molecule raw material vinyl chloride by polyreaction be combined into macromole suspension polymerisation produce, suspension polymerisation by monomer at machinery
Under the effect of stirring or vibration and dispersant, monomer dispersion becomes drop, is typically suspended in water the polymerization process carried out, this side
Method material viscosity is low, and easily heat transfer and control, only need to obtain resin through operations such as simply separating, wash, be dried after polymerization
Product, product is pure, uniform；Described detection zone is coated with the polyclonal antibody of H9N2 subtype avian influenza virus and the inspection that formed
Survey line, described quality control region is coated with the nature controlling line of rabbit anti-mouse igg antibody, and described detection line uses anti-H9N2 subtype avian influenza
The liquid that is coated that Anti-TNF-α bulk concentration is 0.3-0.5mg/mL of virus is coated, and described nature controlling line uses rabbit anti-mouse igg to resist
Bulk concentration is that the liquid that is coated of 1.0-1.2mg/mL is coated；Described neonychium is medical saponified acetate element film, described
Saponified acetate be to be raw material with cellulose, change into, through chemical method, the chemical fibre that acetyl cellulose is made, fast light
Property preferable, expansion and contraction is low, is not affected by chemical substance in composition to be checked.
The preparation method of the test kit of a kind of quick detection H9N2 subtype avian influenza virus is:
Prepared by step one, colloid gold label antigenic solution:
A., after container purified water being cleaned up, add 1wt% aqueous solution of chloraurate and be heated to 100 DEG C, then press 3-5%
Volume ratio dropping add 1.5wt% citric acid three sodium solution, stirring and evenly mixing, react 10-20min, colloidal gold solution is by completely
Become purple, continue to keep 100 DEG C of heating, after colloidal gold solution becomes aubergine, stop heating and be cooled to 25 DEG C, using carbon
Acid potassium solution regulation pH to 8.0-9.0, obtains colloidal gold solution, standby；
B. it is 0.5-with the PBS prepared by H9N2 subtype avian influenza virus antigen diluent to protein content
The diluent of 0.8mg/mL, described PBS component content is 8%BSA, 3% sucrose, 0.8%NaCl and 0.03%
NaN3, then diluent 2400-3000r/min under the conditions of 5-8 DEG C is centrifuged 10-20min, remove precipitation and obtain supernatant, will
Supernatant is added dropwise in the colloidal gold solution of preparation, the antigen that centrifugal segregation is unnecessary, 2400-under the conditions of 5-8 DEG C
3000r/min is centrifuged 30-60min, after standing 8-12h, i.e. obtains the H9N2 subtype avian influenza virus antigen of colloid gold label；
Prepared by step 2, sample pad:
A. by polyethylene glycol 200 12-15g, polyvinylpyrrolidone 15-20g, bovine serum albumin 10-15g, Qu Litong
It is settled to 1000ml with 5mM-10mM phosphate buffer after X-1000 1-1.5ml, polysorbas20 5-8ml mixing, regulates pH value
9.3-9.5 obtains treatment fluid, and described neonychium is soaked in 20-30min in treatment fluid, takes out and seals after 37 DEG C of constant temperature dryings
Deposit, standby；
B. the H9N2 subtype avian influenza virus antigenic solution of colloid gold label is uniformly ejected on glass fibre element film, cold
Lyophilizing is dry, prepares colloid layer gold, then colloid layer gold is bondd with neonychium, i.e. prepares described sample pad.
Step 3, colour developing pad preparation: the polyclonal antibody of anti-H9N2 subtype avian influenza virus is diluted to concentration is 0.3-
0.5mg/mL is coated liquid, is uniformly ejected on nitrocellulose filter and obtains detecting line；Rabbit anti-mouse igg antibody is diluted to concentration
It is coated liquid for 1.0-1.2mg/mL, is uniformly ejected on nitrocellulose filter, obtains nature controlling line, 37 DEG C of freeze-day with constant temperature 3-5h, envelope
Deposit, i.e. obtain described colour developing pad；
Prepared by step 4, test kit: to bonding colour developing pad on base plate, and then bond above colour developing pad absorbent filter,
Colour developing pad mutually overlaps 0.2mm with absorbent filter, between the lower section bond samples pad of colour developing pad, colour developing pad and sample pad mutually
Overlapping 0.2mm, by the width of 5-8mm, the length of 25-30mm carries out cutting cutting, prepares detection card, detection card is placed on plastics
In housing, sample pad is directed at sample collecting district, and colour developing pad is directed at results display area, adds desiccant, loads box body, i.e. makes
Obtain the test kit of a kind of quick detection H9N2 subtype avian influenza virus.
The using method of test kit of the present invention: chicken blood 30 μ l to be detected is dripped in the sample pad of kit sample acquisition zone
On, observe complexion changed situation within 5-10min, the red cell agglutination in chicken blood is suppressed with specific immunity serum, if to be checked
Containing H9N2 subtype avian influenza virus in sample, then the viral-specific antigens of the colloid gold label in sample pad is combined, logical
Cross after the polyclonal antibody of the immunochromatography effect anti-H9N2 subtype avian influenza virus on nitrocellulose filter is combined, can be formed
A macroscopic red detection line, the viral-specific antigens of the colloid gold label be not associated with continue chromatography with and rabbit anti-
Mus IgG antibody also forms macroscopic Article 2 redness nature controlling line after combining；If measuring samples flows without H9N2 hypotype fowl
, the most only there is a red nature controlling line in Influenza Virus antigen；If red nature controlling line does not occurs, then this detection kit lost efficacy.
Having the beneficial effect that of the test kit of the present invention: this test kit is a kind of high specificity, highly sensitive, the fastest
Speed, energy Site Detection, operator are without professional training, and by specification can complete to operate detection kit, utilizes gold colloidal
The principle of immunochromatography, can obtain testing result in 5-10min, it is simple to whether chicken is infected H9N2 subtype avian influenza at normal temperatures
Virus makes quick diagnosis, has good dissemination.
Accompanying drawing explanation
Fig. 1 is the structural representation of the test kit of the quickly detection H9N2 subtype avian influenza virus of the present invention；
Fig. 2 is the detection card structure schematic diagram in the test kit of the quickly detection H9N2 subtype avian influenza virus of the present invention；
Wherein, 1-box body, 2-detects card, 3-sample collecting district, 4-results display area, 5-base plate, 6-absorbent filter, and 7-shows
Color pad, 8-sample pad, 9-detection zone, 10-quality control region, 11-neonychium, 12-colloid layer gold.
Detailed description of the invention
Embodiment 1: the test kit of a kind of quick detection H9N2 subtype avian influenza virus, including box body 1 and detection card 2, institute
The box body stated includes sample collecting district 3 and results display area 4；Described detection card is embedded in box body 1, including base plate 5, water suction
Filter paper 6, colour developing pad 7 and sample pad 8, absorbent filter 6, colour developing pad 7 and sample pad 8 are bonded on base plate 5 successively；Described sample
Pad 8 includes neonychium 11 and colloid layer gold 12, and neonychium 11 is stuck in above colloid layer gold 12, and described colloid layer gold 12 is by being coated with
The glass fibre membrane having the monoclonal antibody of the H9N2 subtype avian influenza virus of colloid gold particle labelling is made, described colour developing pad
7 include that detection zone 9 and quality control region 10, described detection zone 9 are coated with the polyclonal antibody of anti-H9N2 subtype avian influenza virus, described
Quality control region 10 be coated with rabbit anti-mouse igg antibody.
Wherein, described base plate 5 for synthetic resin material prepare, described synthetic resin is macromolecular compound, be by
Low molecule raw material vinyl chloride is combined into what macromole suspension polymerisation produced by polyreaction, and monomer is stirred by suspension polymerisation at machinery
Under the effect mixed or vibrate with dispersant, monomer dispersion becomes drop, is typically suspended in water the polymerization process carried out, this method
Material viscosity is low, easily heat transfer and control, only need to obtain resin produce through operations such as simply separating, wash, be dried after polymerization
Product, product is pure, uniform；Described detection zone 9 is coated with the polyclonal antibody of H9N2 subtype avian influenza virus and the inspection that formed
Survey line, described quality control region 10 is coated with the nature controlling line of rabbit anti-mouse igg antibody, and described detection line uses anti-H9N2 hypotype fowl stream
The Anti-TNF-α bulk concentration of Influenza Virus is that the liquid that is coated of 0.3mg/mL is coated, and described nature controlling line uses rabbit anti-mouse igg antibody
Concentration is that the liquid that is coated of 1.0mg/mL is coated；Described neonychium 11 is medical saponified acetate element film, described soap
Changing acetate fiber is to be raw material with cellulose, changes into, through chemical method, the chemical fibre that acetyl cellulose is made, and light resistance is relatively
Good, expansion and contraction is low, is not affected by chemical substance in composition to be checked.
The preparation method of the test kit of a kind of quick detection H9N2 subtype avian influenza virus is:
Prepared by step one, colloid gold label antigenic solution:
A., after container purified water being cleaned up, add 1wt% aqueous solution of chloraurate and be heated to 100 DEG C, then by 3%
Volume ratio dropping adds 1.5wt% citric acid three sodium solution, stirring and evenly mixing, reacts 10min, and colloidal gold solution is by becoming purple completely
Color, continues to keep 100 DEG C of heating, after colloidal gold solution becomes aubergine, stops heating and is cooled to 25 DEG C, uses potassium carbonate molten
Liquid regulation pH to 8.0, obtains colloidal gold solution, standby；
B. it is 0.5mg/mL with the PBS prepared by H9N2 subtype avian influenza virus antigen diluent to protein content
Diluent, described PBS component content is 8%BSA, 3% sucrose, 0.8%NaCl and 0.03%NaN3, then by dilute
Release liquid 2400r/min under the conditions of 5 DEG C and be centrifuged 10min, remove precipitation and obtain supernatant, supernatant is added dropwise to 1 preparation
Colloidal gold solution in, the antigen that centrifugal segregation is unnecessary, under the conditions of 5 DEG C, 2400r/min is centrifuged 30min, after standing 8h, to obtain final product
H9N2 subtype avian influenza virus antigen to colloid gold label；
Prepared by step 2, sample pad:
A. by polyethylene glycol 200 12g, polyvinylpyrrolidone 15g, bovine serum albumin 10g, vertical logical X-1000 is bent
Being settled to 1000ml with 5mM phosphate buffer after 1ml, polysorbas20 5ml mixing, regulation pH value 9.3 obtains treatment fluid, by institute
The neonychium 11 stated is soaked in 20min in treatment fluid, takes out and seals up for safekeeping after 37 DEG C of constant temperature dryings, standby；
B. the H9N2 subtype avian influenza virus antigenic solution of colloid gold label is uniformly ejected on glass fibre element film, cold
Lyophilizing is dry, prepares colloid layer gold 12, then colloid layer gold 12 is bondd with neonychium 11, i.e. prepares described sample pad 8.
Step 3, colour developing pad 7 preparation: the polyclonal antibody of anti-H9N2 subtype avian influenza virus is diluted to concentration is
0.3mg/mL is coated liquid, is uniformly ejected on nitrocellulose filter and obtains detecting line；Rabbit anti-mouse igg antibody is diluted to concentration
It is coated liquid for 1.0mg/mL, is uniformly ejected on nitrocellulose filter, obtains nature controlling line, 37 DEG C of freeze-day with constant temperature 3h, seal up for safekeeping, to obtain final product
To described colour developing pad 7；
Prepared by step 4, test kit: to bonding colour developing pad 7 on base plate 5, then bonding water suction filter above colour developing pad 7
Paper 6, colour developing pad 7 mutually overlaps 0.2mm with absorbent filter 6, at the lower section bond samples pad 8 of colour developing pad 7, colour developing pad 7 and sample
The most overlapping 0.2mm between pad 8, by the width of 5mm, the length of 25mm carries out cutting cutting, prepares detection card 2, by detection card 2
Being placed in plastic casing, sample pad 8 is directed at sample collecting district 3, and colour developing pad 7 is directed at results display area 4, adds desiccant,
Load box body, i.e. prepare the test kit of a kind of quick detection H9N2 subtype avian influenza virus.
Embodiment 2: the test kit of a kind of quick detection H9N2 subtype avian influenza virus, including box body 1 and detection card 2, institute
The box body stated includes sample collecting district 3 and results display area 4；Described detection card is embedded in box body 1, including base plate 5, water suction
Filter paper 6, colour developing pad 7 and sample pad 8, absorbent filter 6, colour developing pad 7 and sample pad 8 are bonded on base plate 5 successively；Described sample
Pad 8 includes neonychium 11 and colloid layer gold 12, and neonychium 11 is stuck in above colloid layer gold 12, and described colloid layer gold 12 is by being coated with
The glass fibre membrane having the monoclonal antibody of the H9N2 subtype avian influenza virus of colloid gold particle labelling is made, described colour developing pad
7 include that detection zone 9 and quality control region 10, described detection zone 9 are coated with the polyclonal antibody of anti-H9N2 subtype avian influenza virus, described
Quality control region 10 be coated with rabbit anti-mouse igg antibody.
Wherein, described base plate 5 for synthetic resin material prepare, described synthetic resin is macromolecular compound, be by
Low molecule raw material vinyl chloride is combined into what macromole suspension polymerisation produced by polyreaction, and monomer is stirred by suspension polymerisation at machinery
Under the effect mixed or vibrate with dispersant, monomer dispersion becomes drop, is typically suspended in water the polymerization process carried out, this method
Material viscosity is low, easily heat transfer and control, only need to obtain resin produce through operations such as simply separating, wash, be dried after polymerization
Product, product is pure, uniform；Described detection zone 9 is coated with the polyclonal antibody of H9N2 subtype avian influenza virus and the inspection that formed
Survey line, described quality control region 10 is coated with the nature controlling line of rabbit anti-mouse igg antibody, and described detection line uses anti-H9N2 hypotype fowl stream
The Anti-TNF-α bulk concentration of Influenza Virus is that the liquid that is coated of 0.4mg/mL is coated, and described nature controlling line uses rabbit anti-mouse igg antibody
Concentration is that the liquid that is coated of 1.1mg/mL is coated；Described neonychium 11 is medical saponified acetate element film, described soap
Changing acetate fiber is to be raw material with cellulose, changes into, through chemical method, the chemical fibre that acetyl cellulose is made, and light resistance is relatively
Good, expansion and contraction is low, is not affected by chemical substance in composition to be checked.
The preparation method of the test kit of a kind of quick detection H9N2 subtype avian influenza virus is:
Prepared by step one, colloid gold label antigenic solution:
A., after container purified water being cleaned up, add 1wt% aqueous solution of chloraurate and be heated to 100 DEG C, then by 4%
Volume ratio dropping adds 1.5wt% citric acid three sodium solution, stirring and evenly mixing, reacts 15min, and colloidal gold solution is by becoming purple completely
Color, continues to keep 100 DEG C of heating, after colloidal gold solution becomes aubergine, stops heating and is cooled to 25 DEG C, uses potassium carbonate molten
Liquid regulation pH to 8.5, obtains colloidal gold solution, standby；
B. it is 0.65mg/ with the PBS prepared by H9N2 subtype avian influenza virus antigen diluent to protein content
The diluent of mL, described PBS component content is 8%BSA, 3% sucrose, 0.8%NaCl and 0.03%NaN3, then will
Diluent 2700r/min under the conditions of 6.5 DEG C is centrifuged 15min, removes precipitation and obtains supernatant, supernatant is added dropwise to 1
In the colloidal gold solution of preparation, the antigen that centrifugal segregation is unnecessary, under the conditions of 6.5 DEG C, 2700r/min is centrifuged 45min, stands
After 10h, i.e. obtain the H9N2 subtype avian influenza virus antigen of colloid gold label；
Prepared by step 2, sample pad:
A. by polyethylene glycol 200 13.5g, PVP17 .5g, bovine serum albumin 12.5g, vertical logical X-is bent
Being settled to 1000ml with 7.5mM phosphate buffer after 1000 1.25ml, polysorbas20 6.5ml mixing, regulation pH value 9.4 obtains
Treatment fluid, is soaked in 25min in treatment fluid by described neonychium 11, takes out and seal up for safekeeping after 37 DEG C of constant temperature dryings, standby；
B. the H9N2 subtype avian influenza virus antigenic solution of colloid gold label is uniformly ejected on glass fibre element film, cold
Lyophilizing is dry, prepares colloid layer gold 12, then colloid layer gold 12 is bondd with neonychium 11, i.e. prepares described sample pad 8.
Step 3, colour developing pad 7 preparation: the polyclonal antibody of anti-H9N2 subtype avian influenza virus is diluted to concentration is
0.4mg/mL is coated liquid, is uniformly ejected on nitrocellulose filter and obtains detecting line；Rabbit anti-mouse igg antibody is diluted to concentration
It is coated liquid for 1.1mg/mL, is uniformly ejected on nitrocellulose filter, obtains nature controlling line, 37 DEG C of freeze-day with constant temperature 4h, seal up for safekeeping, to obtain final product
To described colour developing pad 7；
Prepared by step 4, test kit: to bonding colour developing pad 7 on base plate 5, then bonding water suction filter above colour developing pad 7
Paper 6, colour developing pad 7 mutually overlaps 0.2mm with absorbent filter 6, at the lower section bond samples pad 8 of colour developing pad 7, colour developing pad 7 and sample
The most overlapping 0.2mm between pad 8, by the width of 6.5mm, the length of 27.5mm carries out cutting cutting, prepares detection card 2, will inspection
Surveying card 2 to be placed in plastic casing, sample pad 8 is directed at sample collecting district 3, and colour developing pad 7 is directed at results display area 4, adds dry
Drying prescription, loads box body, i.e. prepares the test kit of a kind of quick detection H9N2 subtype avian influenza virus.
Embodiment 3: the test kit of a kind of quick detection H9N2 subtype avian influenza virus, including box body 1 and detection card 2, institute
The box body stated includes sample collecting district 3 and results display area 4；Described detection card is embedded in box body 1, including base plate 5, water suction
Filter paper 6, colour developing pad 7 and sample pad 8, absorbent filter 6, colour developing pad 7 and sample pad 8 are bonded on base plate 5 successively；Described sample
Pad 8 includes neonychium 11 and colloid layer gold 12, and neonychium 11 is stuck in above colloid layer gold 12, and described colloid layer gold 12 is by being coated with
The glass fibre membrane having the monoclonal antibody of the H9N2 subtype avian influenza virus of colloid gold particle labelling is made, described colour developing pad
7 include that detection zone 9 and quality control region 10, described detection zone 9 are coated with the polyclonal antibody of anti-H9N2 subtype avian influenza virus, described
Quality control region 10 be coated with rabbit anti-mouse igg antibody.
Wherein, described base plate 5 for synthetic resin material prepare, described synthetic resin is macromolecular compound, be by
Low molecule raw material vinyl chloride is combined into what macromole suspension polymerisation produced by polyreaction, and monomer is stirred by suspension polymerisation at machinery
Under the effect mixed or vibrate with dispersant, monomer dispersion becomes drop, is typically suspended in water the polymerization process carried out, this method
Material viscosity is low, easily heat transfer and control, only need to obtain resin produce through operations such as simply separating, wash, be dried after polymerization
Product, product is pure, uniform；Described detection zone 9 is coated with the polyclonal antibody of H9N2 subtype avian influenza virus and the inspection that formed
Survey line, described quality control region 10 is coated with the nature controlling line of rabbit anti-mouse igg antibody, and described detection line uses anti-H9N2 hypotype fowl stream
The Anti-TNF-α bulk concentration of Influenza Virus is that the liquid that is coated of 0.5mg/mL is coated, and described nature controlling line uses rabbit anti-mouse igg antibody
Concentration is that the liquid that is coated of 1.2mg/mL is coated；Described neonychium 11 is medical saponified acetate element film, described soap
Changing acetate fiber is to be raw material with cellulose, changes into, through chemical method, the chemical fibre that acetyl cellulose is made, and light resistance is relatively
Good, expansion and contraction is low, is not affected by chemical substance in composition to be checked.
The preparation method of the test kit of a kind of quick detection H9N2 subtype avian influenza virus is:
Prepared by step one, colloid gold label antigenic solution:
A., after container purified water being cleaned up, add 1wt% aqueous solution of chloraurate and be heated to 100 DEG C, then by 5%
Volume ratio dropping adds 1.5wt% citric acid three sodium solution, stirring and evenly mixing, reacts 20min, and colloidal gold solution is by becoming purple completely
Color, continues to keep 100 DEG C of heating, after colloidal gold solution becomes aubergine, stops heating and is cooled to 25 DEG C, uses potassium carbonate molten
Liquid regulation pH to 9.0, obtains colloidal gold solution, standby；
B. it is 0.8mg/mL with the PBS prepared by H9N2 subtype avian influenza virus antigen diluent to protein content
Diluent, described PBS component content is 8%BSA, 3% sucrose, 0.8%NaCl and 0.03%NaN3, then by dilute
Release liquid 3000r/min under the conditions of 8 DEG C and be centrifuged 20min, remove precipitation and obtain supernatant, supernatant is added dropwise to 1 preparation
Colloidal gold solution in, the antigen that centrifugal segregation is unnecessary, under the conditions of 8 DEG C, 3000r/min is centrifuged 60min, after standing 12h, i.e.
Obtain the H9N2 subtype avian influenza virus antigen of colloid gold label；
Prepared by step 2, sample pad:
A. by polyethylene glycol 200 15g, polyvinylpyrrolidone 20g, bovine serum albumin 15g, vertical logical X-1000 is bent
Being settled to 1000ml with 10mM phosphate buffer after 1.5ml, polysorbas20 8ml mixing, regulation pH value 9.5 obtains treatment fluid, will
Described neonychium 11 is soaked in 30min in treatment fluid, takes out and seals up for safekeeping after 37 DEG C of constant temperature dryings, standby；
B. the H9N2 subtype avian influenza virus antigenic solution of colloid gold label is uniformly ejected on glass fibre element film, cold
Lyophilizing is dry, prepares colloid layer gold 12, then colloid layer gold 12 is bondd with neonychium 11, i.e. prepares described sample pad 8.
Step 3, colour developing pad 7 preparation: the polyclonal antibody of anti-H9N2 subtype avian influenza virus is diluted to concentration is
0.5mg/mL is coated liquid, is uniformly ejected on nitrocellulose filter and obtains detecting line；Rabbit anti-mouse igg antibody is diluted to concentration
It is coated liquid for 1.2mg/mL, is uniformly ejected on nitrocellulose filter, obtains nature controlling line, 37 DEG C of freeze-day with constant temperature 5h, seal up for safekeeping, to obtain final product
To described colour developing pad 7；
Prepared by step 4, test kit: to bonding colour developing pad 7 on base plate 5, then bonding water suction filter above colour developing pad 7
Paper 6, colour developing pad 7 mutually overlaps 0.2mm with absorbent filter 6, at the lower section bond samples pad 8 of colour developing pad 7, colour developing pad 7 and sample
The most overlapping 0.2mm between pad 8, by the width of 8mm, the length of 30mm carries out cutting cutting, prepares detection card 2, by detection card 2
Being placed in plastic casing, sample pad 8 is directed at sample collecting district 3, and colour developing pad 7 is directed at results display area 4, adds desiccant,
Load box body, i.e. prepare the test kit of a kind of quick detection H9N2 subtype avian influenza virus.
Test kit specific detection
Specific detection: the H9N2 subtype avian influenza virus of inactivation 60 parts is testing sample, respectively with by embodiment 1, reality
Execute example 2, the test kit of embodiment 3 preparation detects, and detection method is as follows: the sample that testing sample is added drop-wise to test kit is adopted
In collection district, it is allowed to along the detection card free diffusing in test kit, observed result in 5-10min.
Result: a red detection line and red nature controlling line, then a result occurs in inactivation H9N2 subtype avian influenza virus
Being judged to the positive, a red nature controlling line only occur, result is judged to feminine gender, if redfree line occurs, loses efficacy, Jing Guoshi
Test and show that the accuracy rate that the H9N2 subtype avian influenza virus of inactivation is detected by test kit prepared by three groups of embodiments is 100%, special
The opposite sex is clearly.
Last it is noted that above example is only in order to illustrate technical scheme, it is not intended to limit；Although
With reference to previous embodiment, the present invention is described in detail, it will be understood by those within the art that: it still may be used
So that the technical scheme described in previous embodiment to be modified, or wherein portion of techniques feature is carried out equivalent；And
These amendments or replacement, do not make the essence of appropriate technical solution depart from spirit and the model of embodiment of the present invention technical scheme
Enclose.
Claims (3)
1. a test kit for quick detection H9N2 subtype avian influenza virus, including box body (1) and detection card (2), its feature exists
In, described box body includes sample collecting district (3) and results display area (4)；Described detection card is embedded in box body (1), bag
Including base plate (5), absorbent filter (6), colour developing pad (7) and sample pad (8), absorbent filter (6), colour developing pad (7) and sample pad (8) depend on
Secondary it is bonded on base plate (5)；Described sample pad (8) includes neonychium (11) and colloid layer gold (12), and neonychium (11) is stuck in
Colloid layer gold (12) top, described colloid layer gold (12) is by the H9N2 subtype avian influenza virus scribbling colloid gold particle labelling
The glass fibre membrane of monoclonal antibody make, described colour developing pad (7) includes detection zone (9) and quality control region (10), described inspection
Surveying district (9) and be coated with the polyclonal antibody of anti-H9N2 subtype avian influenza virus, described quality control region (10) is coated with rabbit anti-mouse igg
Antibody.
The test kit of a kind of quick detection H9N2 subtype avian influenza virus the most as claimed in claim 1, it is characterised in that described
Base plate (5) be synthetic resin material prepare；Described detection zone (9) is coated with the Anti-TNF-α of H9N2 subtype avian influenza virus
Body and the detection line that formed, described quality control region (10) is coated with the nature controlling line of rabbit anti-mouse igg antibody, and described detection line uses
The Anti-TNF-α bulk concentration of anti-H9N2 subtype avian influenza virus is that the liquid that is coated of 0.3-0.5mg/mL is coated, described nature controlling line
The liquid that is coated using rabbit anti-mouse igg antibody concentration to be 1.0-1.2mg/mL is coated；Described neonychium (11) is medical soap
Change cellulose acetate membrane.
The test kit of a kind of quick detection H9N2 subtype avian influenza virus the most as claimed in claim 1, it is characterised in that preparation
Method is:
Prepared by step one, colloid gold label antigenic solution:
A., after container purified water being cleaned up, add 1wt% aqueous solution of chloraurate and be heated to 100 DEG C, then press the body of 3-5%
Long-pending ratio dropping addition 1.5wt% citric acid three sodium solution, stirring and evenly mixing, react 10-20min, colloidal gold solution is by becoming completely
Purple, continues to keep 100 DEG C of heating, after colloidal gold solution becomes aubergine, stops heating and is cooled to 25 DEG C, using potassium carbonate
Solution regulation pH to 8.0-9.0, obtains colloidal gold solution, standby；
B. it is 0.5-0.8mg/mL with the PBS prepared by H9N2 subtype avian influenza virus antigen diluent to protein content
Diluent, described PBS component content is 8%BSA, 3% sucrose, 0.8%NaCl and 0.03%NaN3, then by dilute
Release liquid 2400-3000r/min under the conditions of 5-8 DEG C and be centrifuged 10-20min, remove precipitation and obtain supernatant, supernatant is dropwise added
Entering in the colloidal gold solution prepared to (1), the antigen that centrifugal segregation is unnecessary, under the conditions of 5-8 DEG C, 2400-3000r/min is centrifuged
30-60min, after standing 8-12h, i.e. obtains the H9N2 subtype avian influenza virus antigen of colloid gold label；
Prepared by step 2, sample pad:
A. by polyethylene glycol 200 12-15g, polyvinylpyrrolidone 15-20g, bovine serum albumin 10-15g, vertical logical X-is bent
It is settled to 1000ml with 5mM-10mM phosphate buffer after 1000 1-1.5ml, polysorbas20 5-8ml mixing, regulates pH value
9.3-9.5 obtains treatment fluid, described neonychium (11) is soaked in 20-30min in treatment fluid, takes out in 37 DEG C of constant temperature dryings
After seal up for safekeeping, standby；
B. the H9N2 subtype avian influenza virus antigenic solution of colloid gold label is uniformly ejected on glass fibre element film, freezing dry
Dry, prepare colloid layer gold (12), then colloid layer gold (12) is bondd with neonychium (11), i.e. prepare described sample pad (8).
Prepared by step 3, colour developing pad (7): the polyclonal antibody of anti-H9N2 subtype avian influenza virus is diluted to concentration is 0.3-
0.5mg/mL is coated liquid, is uniformly ejected on nitrocellulose filter and obtains detecting line；Rabbit anti-mouse igg antibody is diluted to concentration
It is coated liquid for 1.0-1.2mg/mL, is uniformly ejected on nitrocellulose filter, obtains nature controlling line, 37 DEG C of freeze-day with constant temperature 3-5h, envelope
Deposit, i.e. obtain described colour developing pad (7)；
Prepared by step 4, test kit: to upper bonding colour developing pad (7) of base plate (5), then in the bonding water suction of the top of colour developing pad (7)
Filter paper (6), colour developing pad (7) and absorbent filter (6) mutually overlap 0.2mm, in lower section bond samples pad (8) of colour developing pad (7), aobvious
The most overlapping 0.2mm between color pad (7) and sample pad (8), by the width of 5-8mm, the length of 25-30mm carries out cutting cutting,
Preparing detection card (2), detection card (2) be placed in plastic casing, sample pad (8) is directed at sample collecting district (3), colour developing pad
(7) it is directed at results display area (4), adds desiccant, load box body, i.e. prepare a kind of quickly detection H9N2 subtype avian influenza sick
The test kit of poison.
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| CN201610528528.5A CN106198970A (en) | 2016-07-06 | 2016-07-06 | A kind of test kit of quick detection H9N2 subtype avian influenza virus |
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| CN201610528528.5A CN106198970A (en) | 2016-07-06 | 2016-07-06 | A kind of test kit of quick detection H9N2 subtype avian influenza virus |
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Cited By (5)
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| CN106478816A (en) * | 2016-12-12 | 2017-03-08 | 青岛蔚蓝生物制品有限公司 | A kind of single-chain antibody of anti-avian influenza H9N2 virus |
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| CN106771245A (en) * | 2016-12-29 | 2017-05-31 | 上海鸣捷生物科技有限公司 | H9 subtype avian influenza virus antigen conjugated magnetic particulates and its preparation method and application |
| KR20220161050A (en) * | 2021-05-28 | 2022-12-06 | 서울대학교산학협력단 | Monoclonal antibody specific to avian influenza virus H9N2 and uses thereof |
| KR102554233B1 (en) | 2021-05-28 | 2023-07-11 | 서울대학교산학협력단 | Monoclonal antibody specific to avian influenza virus H9N2 and uses thereof |
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