CN107589252A - A kind of H5 subtype avian influenza virus fluorescent microsphere detection card and its application - Google Patents
A kind of H5 subtype avian influenza virus fluorescent microsphere detection card and its application Download PDFInfo
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Abstract
A kind of H5 subtype avian influenza virus fluorescent chromatographic test strips, including backing (1), absorb water paper washer (7), cellulose membrane (2), sample pad (4), and it is coated with the fluorescence labeling pad of fluorescent microsphere mark H5 subtype avian influenza virus monoclonal antibody (4A3), wherein, the upper surface of cellulose membrane (2) is from left to right sequentially provided with another plant of H5 subtype avian influenza virus monoclonal antibody (4B6) layer and goat-anti Balb/c mouse immuning ball protein layers, wherein H5 subtype avian influenza virus monoclonal antibody (4B6) layer is provided with detection line (5), goat-anti Balb/c mouse immuning ball proteins layer is provided with control line (6).H5 subtype avian influenza virus fluorescent chromatographic test strips are simple to operate, and practicality is extremely strong, and detection efficiency is higher.
Description
Technical field
The invention belongs to field of virus detection, and in particular to a kind of H5 subtype avian influenza virus fluorescent microsphere detection card and its
Using.
Background technology
Bird flu (Avian influenza, AI) is the birds high degree in contact as caused by orthomyxoviridae family's influenza A
Sexually transmitted disease, there is 16 HA hypotypes and 10 NA hypotypes, and avian influenza virus (Avian influenza virus, AIV) is to support fowl
One of industry destructiveness disease, belong to orthomyxoviridae family's Influenza Virus (Influenza virus), its infection scope is wider, root
According to the difference of pathogenicity, HPAIV and LPAIV can be divided into.Highly pathogenic H5
Subtype avian influenza virus not only causes to seriously endanger to poultry, has a strong impact on international trade, also the health of the mankind is formed seriously
Threaten.Over several years, serious threat avian health to H5N1 subtype avian influenza virus always, and causes huge economic loss.
In recent years, occurs the situation of the H5N1 subtype avian influenza virus infection mankind in succession all over the world, infectious age is up to 50%, prompts
It, which has, causes pandemic potential.H5 hypotypes AIV causes epidemic outbreak in January, 2014 in South Korea first, subsequent rapid seat
Asia, Europe and North America are rolled up；H5N8 subtype highly pathogenic avian influenza epidemic situations have been broken out in October, 2016, Europe again, and
Propagate rapidly, harm is serious.
At present, the detection for AIV mainly passes through pathogen separation and serological test, and these methods are time-consuming, laborious,
Detection in practical application to the AIV of mixed infection has certain limitation.Real-time fluorescence quantitative PCR is by regular-PCR side
Method is combined with fluorescence detection method, can be combined and indicated glimmering with amplification template specificity by being added in PCR reaction systems
The probe of light group, PCR processes are monitored in real time by fluorescence accumulation, as a result can be not required to directly by computer Real Time Observation
Gel electrophoresis is carried out, corresponding viral content can be estimated according to the thresholding of fluorescence curve in sample, realize real-time, quantitative
The purpose of detection, but required detecting instrument is expensive, and laboratory condition is harsh, is not easy to promote in basic unit's Veterinary office.Collaurum is examined
Survey method detection sensitivity is too low, it is easy to causes missing inspection.Therefore be badly in need of want one kind can it is fast and convenient, testing cost is low, detect
The detection of H5 subtype avian influenza virus of efficiency high, high sensitivity has become a big problem in industry.
The content of the invention
Immuno-chromatographic assay technology based on fluorescent microsphere, which compares collaurum detection, can improve 10 times -100 times, can realize
Site Detection, and can enough realize super sensitivity detection, are the inexorable trends for substituting the low Sensitive Detection of bird flu collaurum.Pass through formulation
Avian influenza virus line fluorescent Site Detection standard that is advanced, reaching the international leading level, can improve disposal bird flu conscientiously
The ability of Emergent Public Events, it will influence to be preferably minimized limit caused by bird flu Emergent Public Events.
That is, it is an object of the invention to provide a kind of H5 subtype avian influenza virus fluorescent chromatographic test strips, including backing
(1), absorb water paper washer (7), cellulose membrane (2), sample pad (4) and be coated with fluorescent microsphere mark H5 subtype avian influenza virus
The fluorescence labeling pad of monoclonal antibody (4A3), cellulose membrane (2) are located on backing (1), and the right side of fluorescence labeling pad (3) is located at
The left side of cellulose membrane (2) upper surface, cellulose membrane (2) is reached outside on the left of fluorescence labeling pad (3), a left side for sample pad (4)
Side is located on fluorescence labeling pad (3), and fluorescence labeling pad (3) is reached on the right side of sample pad (4) outside, the left side of water suction paper washer (7)
Cellulose membrane (2) is reached on the right side of the right side of cellulose membrane (2) upper surface, water suction paper washer (7) outside；
Wherein, the upper surface of cellulose membrane (2) is from left to right sequentially provided with another plant of H5 subtype avian influenza virus monoclonal
Antibody (4B6) layer and goat-anti Balb/c mouse immuning ball protein layers, wherein H5 subtype avian influenza virus monoclonal antibody (4B6) layer
Detection line (5) is provided with, goat-anti Balb/c mouse immuning ball proteins layer is provided with control line (6).
Preferably, in H5 subtype avian influenza virus fluorescent chromatographic test strips of the present invention, the cellulose membrane (2) is
Nitrocellulose filter.
Preferably, in H5 subtype avian influenza virus fluorescent chromatographic test strips of the present invention, the nitrocellulose filter
Model nitrocellulose filter 140.
Preferably, in H5 subtype avian influenza virus fluorescent chromatographic test strips of the present invention, the cellulose membrane (2)
Thickness is 0.2mm, width 3.5mm.
Preferably, in H5 subtype avian influenza virus fluorescent chromatographic test strips of the present invention, the fluorescent microsphere mark
In H5 subtype avian influenza virus antibody fluorescent microsphere be average diameter be 110nm, the fluorescent microsphere of carboxyl modified.
Preferably, in H5 subtype avian influenza virus fluorescent chromatographic test strips of the present invention, the fluorescent microsphere and H5
The mass ratio of subtype avian influenza virus hemagglutinin monoclonal antibody is 5:0.35.
Preferably, in H5 subtype avian influenza virus fluorescent chromatographic test strips of the present invention, the detection wire spraying H5
The package amount of subtype avian influenza virus antibody is the μ l/cm of 0.5 μ l/cm~1.5；Nature controlling line is used for the bag for drawing the sheep anti mouse secondary antibody of film
Measured as the μ l/cm of 0.5 μ l/cm~1.5.
Preferably, in H5 subtype avian influenza virus fluorescent chromatographic test strips of the present invention, the detection line is used to draw
The coating concentration of the avian influenza virus monoclonal antibody (4B6) of film is 1.5mg/ml~2.1mg/ml；Fluorescent microsphere and H5 hypotypes
The discharge rate of avian flu virus hemagglutinin monoclonal antibody (4A3) conjugate is the μ l/cm of 2.0 μ l/cm~2.5.
Preferably, in H5 subtype avian influenza virus fluorescent chromatographic test strips of the present invention, the nature controlling line, which sprays, to be used for
The concentration for drawing the sheep anti mouse secondary antibody of film is 0.5mg/ml~2mg/ml
It is sub- in detection H5 another object of the present invention is to provide above-mentioned H5 subtype avian influenza virus fluorescent chromatographic test strips
Application in type avian influenza virus, i.e., sample is carried out using H5 subtype avian influenza virus fluorescent chromatographic test strips as described above
Detection：
Such as there is no band in control line (6), then it is invalid to detect；Such as there is band in control line (6), detection line (5) does not go out
Existing band, then be feminine gender；Such as there is band in control line (6), band occurs in detection line (5), then is the positive.
Compared with prior art, the present invention has advantages below to the present invention：
Fluorescent chromatographic test strips of the present invention are when in use, it is only necessary to glue the oral cavity of animal to be detected or cloaca
Liquor, tissue fluid are added dropwise in sample pad, and when sample has H5 subtype avian influenza virus, then H5 subtype avian influenza virus is anti-
The former fluorescence labeling H5 AIV monoclonal antibodies (4A3) with fluorescence labeling pad, which react, generates H5 subtype avian influenza virus-fluorescence mark
Remember the compound of H5 AIV monoclonal antibodies, the compound of H5 subtype avian influenza virus-fluorescence labeling H5 AIV monoclonal antibodies (4A3) moves right
During to H5 AIV monoclonal antibodies (4B6) layer, then it is mono- to form H5 AIV monoclonal antibodies (4B6)-H5 subtype avian influenza virus-fluorescence labeling H5 AIV
The compound of anti-(4A3), and be formed about in detection line exciting fluorescent bands, while uncombined fluorescence labeling H5 AIV monoclonal antibodies
Move right, and generation goat-anti Balb/c mouse immuning ball proteins-fluorescence mark is reacted with goat-anti Balb/c mouse immuning ball proteins
The compound of anti-H5 AIV monoclonal antibodies (4A3) is remembered, so as to being formed about exciting fluorescent bands in control line, i.e., with specific fluorescent layer
When analysing analyzer detection, when on the liquid in detection line and control line while when there are fluorescent bands, then illustrating animal to be detected
Simple to operate containing H5 subtype avian influenza virus, practicality is extremely strong, and detection efficiency is higher.
Brief description of the drawings
The H5 subtype avian influenza virus fluorescent chromatographic detection card that Fig. 1 is the present invention is developed and application technology route map；
Fig. 2 is that H5 subtype avian influenza virus fluorescent chromatographic of the present invention detects card vertical section and front schematic view；
Fig. 3 is the fluorescent chromatographic detection card detection reaction schematic diagram of the present invention；
Fig. 4 is the test result signal of the H5 subtype avian influenza virus fluorescent microsphere detection card in one embodiment of the invention
Figure；
Fig. 5 is the H5 subtype avian influenza virus fluoroscopic examination card test result figures in one embodiment of the invention.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings：
With reference to figure 2, fluorescent chromatographic test strips structure of the present invention include backing 1, water suction paper washer 7, cellulose membrane 2,
Sample pad 4 and the fluorescence labeling pad 3 containing fluorescence labeling H5 AIV monoclonal antibodies (4A3), cellulose membrane 2 is located on backing 1, glimmering
The right side of signal pad 3 is located at the left side of the upper surface of cellulose membrane 2, and the left side of fluorescence labeling pad 3 is reached outside cellulose membrane 2,
The left side of sample pad 4 is located on fluorescence labeling pad 3, and the right side of sample pad 4 is reached outside fluorescence labeling pad 3, a left side for the paper washer 7 that absorbs water
Side is located at the right side of the upper surface of cellulose membrane 2, and the right side of water suction paper washer 7 is reached outside cellulose membrane 2；The upper table of cellulose membrane 2
Face is from left to right sequentially provided with H5 AIV monoclonal antibodies (4B6) layer and goat-anti Balb/c mouse immuning ball protein layers, wherein H5 AIV are mono-
Anti- (4B6) layer is provided with detection line 5, and goat-anti Balb/c mouse immuning ball proteins layer is provided with control line 6.
It should be noted that the cellulose membrane 2 is nitrocellulose filter, the thickness of cellulose membrane 2 is 0.2cm, fiber
The width of plain film 2 is 3.5mm.
The present invention detection process be：
Only need the mucus of animal to be detected or tissue fluid being added dropwise in sample pad 4, when sample pad 4 has H5 hypotypes fowl stream
During Influenza Virus, then the antigen of H5 subtype avian influenza virus occurs with the fluorescence labeling H5 AIV monoclonal antibodies (4A3) on fluorescence labeling pad 3
The compound of reaction generation H5 subtype avian influenza virus-fluorescence labeling H5 AIV monoclonal antibodies (4A3), while in the work of water suction paper washer 7
Under, the compound of H5 subtype avian influenza virus-fluorescence labeling H5 AIV monoclonal antibodies (4A3) moves right to H5 AIV monoclonal antibodies
During (4B6) layer, then answering for H5 AIV monoclonal antibodies (4B6)-H5 subtype avian influenza virus-fluorescence labeling H5 AIV monoclonal antibody (4A3) is formed
Compound, and fluorescent bands are formed about in detection line 5, while uncombined fluorescence labeling H5 AIV monoclonal antibodies move right, and with
The goat-anti Balb/c mouse immuning ball proteins reaction generation goat-anti Balb/c mouse immuning ball proteins-anti-H5 AIV monoclonal antibodies of fluorescence labeling
The compound of (4A3), so as in control line 6 be formed about fluorescent bands, when being detected with fluorescence analyser, when detection line 5 and right
During according to fluorescent bands occur simultaneously on line 6, then illustrate to contain H5 subtype avian influenza virus on the liquid of animal to be detected；Work as control
When not occurring fluorescent bands within line 6, then illustrate not containing H5 subtype avian influenza virus on the liquid of animal to be detected, when
When control line 6 is without display fluorescent bands, then illustrates fluorescent chromatographic test strips failure of the present invention or operate incorrect.
Below in conjunction with the embodiment in the present invention, the technical scheme in the present invention is clearly and completely described.It is aobvious
So, described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on the reality in the present invention
Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, is all belonged to
In the scope of protection of the invention.
The fluorescent microsphere of embodiment 1 marks the preparation of avian influenza virus antibody
The use of average diameter is 110nm, the fluorescent microsphere of carboxyl modified (Bangs Laboratories, Inc. companies, production
Product catalog number (Cat.No.) is FC02F/10930), and monoclonal antibody (4A3 and the 4B6) (purchase of anti-H5 subtype avian influenza virus hemagglutinin
From animal medicine institute of Yangzhou University, infectious disease and prevention animal doctor key lab), fluorescent microsphere mark is prepared by the following method
Remember avian influenza virus marking antibody：
Using average diameter as 110nm, the fluorescent microsphere of carboxyl modified, the monoclonal of anti-H5 subtype avian influenza virus hemagglutinin
Antibody, fluorescent microsphere mark avian influenza virus marking antibody is prepared by the following method：
After taking the fluorescent microsphere of 5mg above-mentioned carboxyl modified to be washed and centrifuged with MES buffer solutions (0.1M, pH4.7), use
1ml MES buffer solutions (0.1M, pH4.7) are resuspended, and add 1- ethyls-(3- dimethylaminopropyls) carbodiimide (EDC) to end
Concentration is 5mM, adds NHS (N- hydroxysuccinimides) to final concentration of 10mM, and room temperature lucifuge, reaction half an hour is activated
The fluorescent microsphere of carboxyl modified afterwards.
The fluorescent microsphere of carboxyl modified after washing the activation with 50mM pH8.5 borate buffer solution, takes 0.35mg is above-mentioned to treat
The fluorescent microsphere of carboxyl modified is mixed into 50mMpH8.5 after mark H5 subtype avian influenza virus antibody (4A3) and the above-mentioned activation of 5mg
Borate buffer solution in fully mix.Reacted 2 hours under room temperature lucifuge, allow antibody and fluorescent microsphere to form stable covalent peptide bonds
With reference to obtaining the conjugate of fluorescent microsphere and H5 subtype avian influenza virus antibody.After reaction terminates, final concentration of 1% (matter is added
Amount percentage composition) BSA solution residual activity carboxyl site on the conjugate of fluorescent microsphere and avian influenza virus antibody is carried out
Closing, room temperature lucifuge are reacted 0.5 hour.After the completion of, washed with pH5.4 0.02M PBSs, be resuspended that to obtain 5mg/ml glimmering
Light microballoon mark avian influenza virus antibody liquid, 4 DEG C of preservations are stand-by.
The preparation of the H5 subtype avian influenza virus fluoroscopic examination cards of embodiment 2
The gained fluorescent microsphere of embodiment 1 is marked into H5 subtype avian influenza virus antibody-solutions, using BioDot XYZ3050
It is sprayed into treated glass fibre element film according to certain discharge rate and (is purchased from waterman and Schleicher＆ by spray membranous system
Schuell companies), preparation forms fluorescence antibody pad, is then moved it into grid tray in a vacuum drying chamber, very
Sky dries 2h, adds drier or discoloration silica gel, is sealed stand-by in 4 DEG C of refrigerators.
The scope for selecting the i.e. spray film amount of detection line and the coating volume of nature controlling line is 0.5 μ l/cm-1.5 μ l/cm, according to warp
Test selection appropriate proteins coating concentration, with automatic Membrane jetter spray detection line, spray film amount be respectively 0.5 μ l/cm, 0.75 μ l/cm,
1.0 μ l/cm, 1.5 μ l/cm, compare the line effect obtained by with different spray film amounts.
H5 subtype avian influenza virus antibody (4B6) is diluted to by various concentrations using antigen, secondary antibody dilution：1.5mg/
Ml, 1.8mg/ml, 2.1mg/ml, carry out detection line coating.Above-mentioned three kinds of Concentration Testings are being coated with automatic Membrane jetter
The nitrocellulose filter of line combines with the pad of different discharge rates, prepares test strips sample and pastes system successively with other auxiliary materials
It is used to detect into quick measuring card and tests.
It is right after bird flue virus H 5 N 1 subtype AI is tested into antigen diluent using the 0.02M PBSs that pH is 7.4
Quick measuring card carries out test experience.
With determine H5 subtype avian influenza virus antibody (4B6) coating concentration and fluorescence labeling conjugate discharge rate basis
On, sheep anti mouse secondary antibody is diluted to by various concentrations using antigen, secondary antibody dilution：2mg/ml、1.5mg/ml、1mg/ml、
0.5mg/ml, nature controlling line coating is carried out, by detecting H5 subtype avian influenza cell culture and virus, determine that nature controlling line is coated with concentration.
Using antigen, secondary antibody dilution, sheep anti mouse secondary antibody and H5 subtype avian influenza virus monoclonal antibody (4B6) are prepared
For certain density solution, membranous system is sprayed by two anti-zoned film of sheep anti mouse to coated film (cellulose nitrate from BioDot XYZ3050
Plain film) nature controlling line (C lines) position, H5 subtype avian influenza virus monoclonal antibody (4B6) is drawn into film to detection line (T lines) position
Put, fluorescent microsphere is marked to the antibody-solutions of H5 subtype avian influenza virus, treated glass fibers are sprayed into according to certain discharge rate
Tie up on plain film, the dried for standby after the drying plant that relative humidity is less than 30% carries out dehumidifier 24 hours.
Dried coated film, the above-mentioned sample with detection line and nature controlling line in 100,000 grades of cleanings and the workshop dried
After product pad, adsorptive pads, backboard carry out collocation assembling, the Paperboard cutting that will be posted using BioDot CM4000 cutting systems, dress
It is stand-by to enter detection intermediate plate.
Experimental result：
The determination result of 1 H5 subtype avian influenza virus fluoroscopic examination cards detection line, nature controlling line coating volume
Rule of thumb selection concentration is with concentration for 2.1mg/ml H5 subtype avian influenza virus monoclonal antibody (4B6)
1.5mg/ml sheep anti mouse secondary antibody is respectively according to coating μ l/cm of volume 0.5,0.75 μ l/cm, 1.0 μ l/cm and 1.5 μ l/cm
Film is drawn, its effect of ruling is as shown in table 1：
The determination of the detection line of table 1, nature controlling line coating volume
Remarks：+ represent that band is clear, concentrates, indiffusion；- represent that band color is not concentrated, be unintelligible, having diffusion；±
Represent in+and-between.
As a result show, when detection line is 1.0 μ l/cm with nature controlling line coating volume, institute's scribing edge is neat, lines essence
Carefully, raw material and is saved the most.
2 H5 subtype avian influenza virus fluoroscopic examination cards detection lines are coated with the determination knot of concentration and pad discharge rate volume
Fruit
Testing experiment is carried out to quick measuring card by the sample of preparation, test result refers to table 2.
The antibody of table 2 is coated with the determination of concentration and discharge rate
Remarks：+ represent that band is clear, concentrates, indiffusion；- represent that band color is not concentrated, be unintelligible, having diffusion；±
Represent in+and-between.
As a result show, when detection line coating concentration is 2.1mg/ml and pad discharge rate volume is 2.3 μ l/cm, colour developing
Band is clear, concentrates.
3 H5 subtype avian influenza virus fluoroscopic examination cards nature controlling lines are coated with the determination result of concentration
Testing experiment is carried out to quick measuring card using H5 subtype avian influenza cell culture and virus samples, test result refers to table 3.
The nature controlling line of table 3 is coated with the determination of concentration
Remarks：+ represent that band is clear, concentrates, indiffusion；- represent that band color is not concentrated, be unintelligible, having diffusion；±
Represent in+and-between.
Test result indicates that coating concentration 1mg/ml, develop the color band in H5 subtype avian influenza cell culture and virus samples
Clearly, concentrate.
Accordingly, it is determined that detection card detection line, nature controlling line coating concentration and pad discharge rate volume, detection line (T lines) H5
Subtype avian influenza virus MAb concentration 2.1mg/ml, nature controlling line (C lines) sheep anti mouse secondary antibody concentration 1mg/ml, pad fowl
The μ l/cm of Antibody of Influenza solution discharge rate volume 2.3.Example 3 below~4 are with the H5 subtype avian influenza virus fluorescence of the determination
Detection card carries out application experiment.
The application test of the H5 subtype avian influenza virus fluoroscopic examination cards of embodiment 3
H5 hypotypes AIV positive serum and negative serum, avian influenza virus H7N9 antigens, H7 hypotypes antigen, H5N1 (Re-
6) antigen, H5N2 antigens, H9N2 antigens, H1N1 antigens, H3N2 antigens, ndv antigen, IBDV antigens, IBV antigens, ILTV antigens
It is purchased from Harbin veterinary institute National Animal engineering center.
With the viral antigen or positive of cell culture, H5 subtype avian influenza virus fluorescence prepared by embodiment 2 is examined
Survey card and carry out result test, its result is judged as shown in figure 4, wherein, not having band in nature controlling line, then it is invalid to detect；In Quality Control
There is band in line, and detection line does not occur band, then is feminine gender；There is band in nature controlling line, band occurs in detection line, then is sun
Property.
Test result is as shown in Figure 5：(p1, p2 are the strain of H5 subtype avian influenza virus).
The performance test of the H5 subtype avian influenza virus fluoroscopic examination cards of embodiment 4
H5 hypotypes AIV positive serum and negative serum, avian influenza virus H7N9 antigens, H7 hypotypes antigen, H5N1 (Re-
6) antigen, H5N2 antigens, H9N2 antigens, H1N1 antigens, H3N2 antigens, ndv antigen, IBDV antigens, IBV antigens, ILTV antigens
It is purchased from Harbin veterinary institute National Animal engineering center.
1st, H5 subtype avian influenza virus fluoroscopic examination card sensitivity result
H5N1 standard antigens are subjected to continuous doubling dilution, the H5 subtype avian influenza virus established using embodiment 2
Fluoroscopic examination card is detected, and its testing result is as shown in table 4 below.
The testing result of the H5 subtype avian influenza virus fluoroscopic examination card difference sample concentrations of table 4
The H5N1 subtype influenzas of H5 subtype avian influenza virus fluoroscopic examination card examination criterias can be drawn from testing result
The sensitivity of virus is 0.031HAunit.
2nd, H5 subtype avian influenza virus fluoroscopic examination card specific outcome
Using the H5 subtype avian influenza virus fluoroscopic examination cards developed, to cause the respiratory tract syndrome of people related and with
Virus is detected similar in influenza, testing result all feminine genders as shown in table 6.It is close to influenza and cause poultry respiratory
The virus of syndrome is detected, testing result also all feminine genders.Detection anima, including the Nasopharyngeal swabs of normal person,
Sputum, the cotton swab of normal poultry, normal poultry organization, testing result total negative.Show established method not by base
The influence of matter, and testing result high specificity.Its testing result is as shown in table 5 below.The H5 hypotypes fowl prepared according to this method is flowed
Influenza Virus fluoroscopic examination card can be used to detect H5 subtype avian influenza virus, with common Respirovirus IBV, reovirus,
There is no cross reaction between NDV.
The H5 subtype avian influenza virus fluoroscopic examination card cross reaction testing results of table 5
| Virus subtype | Testing result |
| H1N1 | - |
| H3N2 | - |
| H5N1 | + |
| H5N2 | + |
| H7 | - |
| H7N9 | - |
| H9N2 | - |
| NDV | - |
| RSV | - |
| Parainfluenza virus | - |
| IBDV | - |
| IBV | - |
| ILTV | - |
3rd, the small-scale field test results of H5 subtype avian influenzas disease fluoroscopic examination card
To gathering 200 parts of field test sample, the detection of H5 subtype avian influenza virus fluoroscopic examination cards is all carried out simultaneously
Detected with fluorescence RT-PCR, both testing results are consistent, show the practical of institute's method for building up.
The H5 subtype avian influenza virus of table 6 detects card detection method and RT-PCR detection method comparing results
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of H5 subtype avian influenza virus fluorescent chromatographic test strips, including backing (1), water suction paper washer (7), cellulose membrane (2),
Sample pad (4) and the fluorescence labeling pad for being coated with fluorescent microsphere mark H5 subtype avian influenza virus monoclonal antibody (4A3),
Cellulose membrane (2) is located on backing (1), and the right side of fluorescence labeling pad (3) is located at the left side of cellulose membrane (2) upper surface, fluorescence
Cellulose membrane (2) is reached on the left of label pad (3) outside, the left side of sample pad (4) is located on fluorescence labeling pad (3), sample pad
(4) fluorescence labeling pad (3) is reached on the right side of outside, the left side of water suction paper washer (7) is located at the right side of cellulose membrane (2) upper surface,
Cellulose membrane (2) is reached on the right side of water suction paper washer (7) outside；
Wherein, the upper surface of cellulose membrane (2) is from left to right sequentially provided with another plant of H5 subtype avian influenza virus monoclonal antibody
(4B6) layer and goat-anti Balb/c mouse immuning ball protein layers, wherein H5 subtype avian influenza virus monoclonal antibody (4B6) layer are provided with detection
Line (5), goat-anti Balb/c mouse immuning ball proteins layer are provided with control line (6).
2. H5 subtype avian influenza virus fluorescent chromatographic test strips according to claim 1, it is characterised in that the cellulose
Film (2) is nitrocellulose filter.
3. H5 subtype avian influenza virus fluorescent chromatographic test strips according to claim 2, it is characterised in that the nitric acid is fine
Tie up the model nitrocellulose filter 140 of plain film.
4. H5 subtype avian influenza virus fluorescent chromatographic test strips according to claim 2, it is characterised in that the cellulose
The thickness of film (2) is 0.2mm, width 3.5mm.
5. H5 subtype avian influenza virus fluorescent chromatographic test strips according to claim 1, it is characterised in that the fluorescence is micro-
In ball mark H5 subtype avian influenza virus monoclonal antibody (4A3) fluorescent microsphere be average diameter be 110nm, carboxyl modified it is glimmering
Light microballoon.
6. H5 subtype avian influenza virus fluorescent chromatographic test strips according to claim 1, it is characterised in that the fluorescence is micro-
Ball and the mass ratio of H5 subtype avian influenza virus hemagglutinin monoclonal antibodies (4A3) are 5:0.35.
7. H5 subtype avian influenza virus fluorescent chromatographic test strips according to claim 1, it is characterised in that the detection line
The package amount of avian influenza virus monoclonal antibody (4B6) for drawing film is the μ l/cm of 0.5 μ l/cm~1.5；Nature controlling line is used to draw
The package amount of the sheep anti mouse secondary antibody of film is the μ l/cm of 0.5 μ l/cm~1.5.
8. H5 subtype avian influenza virus fluorescent chromatographic test strips according to claim 1, it is characterised in that the detection line
The coating concentration of avian influenza virus monoclonal antibody (4B6) for drawing film is 1.5mg/ml~2.1mg/ml；Fluorescent microsphere with
The discharge rate of H5 subtype avian influenza virus hemagglutinin monoclonal antibody (4A3) conjugate is the μ l/cm of 2.0 μ l/cm~2.5.
9. H5 subtype avian influenza virus fluorescent chromatographic test strips according to claim 1, it is characterised in that the nature controlling line
The concentration of sheep anti mouse secondary antibody for drawing film is 0.5mg/ml~2mg/ml.
10. a kind of application of H5 subtype avian influenza virus fluorescent chromatographic test strips in H5 subtype avian influenza virus is detected, it is special
Sign is that usage right requires that the H5 subtype avian influenza virus fluorescent chromatographic test strips described in 1~9 any one are carried out to sample
Detection：
Such as there is no band in control line (6), then it is invalid to detect；Such as there is band in control line (6), detection line (5) does not occur bar
Band, then it is feminine gender；Such as there is band in control line (6), band occurs in detection line (5), then is the positive.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108107206A (en) * | 2017-11-28 | 2018-06-01 | 清华大学深圳研究生院 | The detection kit of A type H5 subtype influenza virus and its application |
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