CN1996021A - Enzyme-linked immunologic adsorption test method for identification and diagnosis of chicken infected with avian influenza - Google Patents
Enzyme-linked immunologic adsorption test method for identification and diagnosis of chicken infected with avian influenza Download PDFInfo
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Abstract
The invention relates to the enzyme combined immune absorbing testing for diagnosing bird flu affected chicks. It expands H9N2 bird flu virus, acquiring target section to connect with the carrier to restructure the expressing particle, injecting into bacillus coli expression to get a conjugated protein which is purified to get H9N2 bird flu virus NS1 protein to be antibody wrapping enzyme reaction plate, to build the antibody level to be reacted to inspect of the serum, getting the marginal value and least sample number of the enzyme combined immune adhesive test through statistics, and finally based on the average OD value to identify the bird flu affection status of the overall poultry. Through overall diagnosis, it alleviates nonspecific reaction, provides scientific proof for timely diagnosis and prevention of bird flu.
Description
Technical field
What the present invention relates to is a kind of method of technical field of bioengineering, specifically is the chicken infected enzyme-linked immunologic adsorption test method of a kind of antidiastole bird flu.
Background technology
Bird flu, especially HPAI (highly pathogenic bird flu) have very big harm to the bird aquaculture.One of effective method of controlling bird flu is vaccine inoculation at present, and an index of effective evaluation immune effect is exactly the antibody horizontal after the detection immunity, and therefore effectively the antibody of the antibody of differentiation natural infection generation and immunity inoculation generation is extremely important.The method of traditional serodiagnosis bird flu has HA-HI (blood clotting and hemagglutination-inhibition test), AGP (agar immunodiffusion), microneutralization and NIF (neuraminidase inhibition test), because the antibody that these methods detected, no matter be that vaccine inoculation or the wild poison of infection all can produce, therefore these detection methods can not truly reflect the actual infection conditions of bird flu, bring great difficulty to prevention and control of fowl influenza.
Find through literature search prior art, Wang Zelin etc. deliver in " Chinese science and technology " (2006 213 volume 14-17 page or leaf) is entitled as " non-structural protein NS 1-enzyme linked immunosorbent assay detects bird flu wild virus infection antibody " paper, and Tao Yang waits the paper of delivering at " Chinese animal doctor's science and technology " (2005 25 volume 585-589 pages or leaves) that is entitled as " prokaryotic expression of H5N1 subtype avian influenza virus NS1 albumen and the foundation of enzyme linked immunosorbent assay detection method thereof ", all to utilizing bird flu non-structural protein (NS1 albumen) as envelope antigen, by the method for enzyme linked immunosorbent assay, the associated antibodies IgG level at NS1 albumen has relevant report in the detection chicken serum on individual level.But on the one hand, NS1 albumen is a kind of poor antigen, is difficult for excitating organism and produces high titer antibody.The chicken group who promptly is natural infection may not can on the Different Individual level produces very high antibody titer; On the other hand, because present used inactivated avian influenza vaccine is all made of the chicken embryo, in the process of chicken embryo propagative viruses, the generation of NS1 albumen is also arranged, in the oil emulsion inactivated vaccine of application chick embryo allantoic liquid preparation NS1 albumen is just arranged, therefore particularly repeatedly there is a certain amount of antibody at NS1 albumen in vaccinated flock among the chicken group of immunity probably, that is to say on individual level, the NS1 antibody horizontal of the chicken of natural infection avian influenza virus may be not high, or the NS1 antibody horizontal of immunity inoculation chicken may be than higher, so the infection conditions of antidiastole bird flu often has very big deviation on the individual level.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of population level antidiastole bird flu chicken infected enzyme-linked immunologic adsorption test method, make it utilize different at natural infection chicken group and immunity inoculation chicken group content of bird flu NS1 protein antibodies on the population level, method by enzyme linked immunosorbent assay detects in the chicken serum and the antigen reactive IgG antibody level of NS1, by critical value and the minimum animal used as test number of determining that bird flu sero-enzyme linked immunosorbent adsorption test yin and yang attribute is judged, from the wild virus infection of population level antidiastole chicken group bird flu.
The present invention is achieved by the following technical solutions, and concrete steps of the present invention comprise:
1. the gene by inverse transcription polymerase chain reaction amplification H9N2 type avian influenza virus NS1 albumen;
2. the gene of gained H9N2 type avian influenza virus NS1 albumen of 1. step being increased is cut by enzyme and is inserted among the vector plasmid pET-32a (+), construct prokaryotic expression plasmid, the plasmid that builds is changed in the e. coli bl21, IPTG (isopropyl sulfydryl galactoside) abduction delivering, expression product is the fusion of molecular weight 44.2KD through polyacrylamide gel electrophoresis and protein immunoblotting experimental identification;
3. with step 2. the gained fusion as antigen coated enzyme reaction plate, set up the method for enzyme linked immunosorbent assay, determine optimum reaction condition, the best bag that comprises antigen is by the optimum diluting multiple of the optimum diluting multiple of concentration, antibody, antigen-antibody optimum reacting time, ELIAS secondary antibody and the optimum reacting time of ELIAS secondary antibody;
4. the method for the enzyme linked immunosorbent assay of 3. setting up according to step detects negative serum, the bird flu H9N2 inactivated vaccine inoculation group chicken serum of bird flu H9N2 infected group chicken serum, the non-immune chicken of 30 ages in days;
5. according to statistical method testing result is analyzed, determined the critical value and the minimum sample number of the enzyme linked immunosorbent assay yin and yang attribute judgement of bird flu serum;
6. according to the critical value of serum sample enzyme linked immunosorbent assay yin and yang attribute judgement and the minimum detection sample number of regulation, according to the size of the enzyme linked immunosorbent assay mean light absorbency of chicken group serum sample, the chicken group that is defined as that mean light absorbency is higher than critical value has infected the wild poison of bird flu.
The critical value that described bird flu sero-enzyme linked immunosorbent adsorption test yin and yang attribute is judged, be after the negative serum to 70 parts of bird flu H9N2 infected group chicken serums, 30 part of 30 non-immune chicken of age in days, 95 parts of bird flu H9N2 inactivated vaccine inoculation group chicken serums carry out statistical analysis, the mean value of getting the negative serum of the non-immune chicken of 30 ages in days adds the critical value that three standard deviations are judged as bird flu serum yin and yang attribute.
The critical value that described bird flu sero-enzyme linked immunosorbent adsorption test yin and yang attribute is judged is 0.5.
Described minimum sample number is by statistical analysis, and the regulation enzyme linked immunosorbent assay determines whether chicken group integral body infects the number of the needed minimum chicken of avian influenza virus.Minimum sample number is 30.
Described optimum reaction condition is: the best bag of antigen is that the optimum diluting multiple of 40 μ g/mL, antibody is that 1: 200, antigen-antibody optimum reacting time are that the optimum diluting multiple of 1h, ELIAS secondary antibody is that the optimum reacting time of 1: 10000 and ELIAS secondary antibody is 1h by concentration.
Described antigen is through the H9N2 of escherichia coli prokaryotic expression type avian influenza virus NS1 albumen, is coated on the mensuration of carrying out antibody on the solid phase carrier, and blood serum sample to be measured is carried out qualitative analysis.
Enzyme linked immunosorbent assay is utilized the antigen or the antibody of enzyme labeling, the antigen that carries out on solid phase carrier or the mensuration of antibody.The basis of enzyme linked immunosorbent assay is the enzyme labeling of immobilization and the antigen or the antibody of antigen or antibody.The antigen or the antibody that are combined in surface of solid phase carriers still keep its immunologic competence, and the antigen of enzyme labeling or antibody had both kept its immunologic competence, kept the activity of enzyme again.When measuring, examined sample (measuring wherein antibody or antigen) and the antigen or the antibody of surface of solid phase carriers and reacted.With the method for washing the antigen antibody complex that forms on the solid phase carrier and other materials in the liquid are separated.Add the antigen or the antibody of enzyme labeling again, also be combined on the solid phase carrier by reaction.The amount of examined object matter is certain ratio in enzyme amount on the solid phase and the sample at this moment.After adding the substrate of enzyme reaction, substrate is become coloured product by enzymatic, and the amount of product is directly related with the amount of examined object matter in the sample, so can carry out qualitative or quantitative test according to the depth of the color that presents.The present invention be with avian influenza virus NS1 albumen as antigen coated enzyme reaction plate, the test serum sample is diluted and antigen-reactive according to a certain percentage, add ELIAS secondary antibody, add the substrate colour developing at last, come result of determination according to the OD value of measuring the colour developing sample.
Described H9N2 type avian influenza virus is meant that influenza A virus hemagglutinin (H) and neuraminidase (N) are divided into 15 H types and 9 N types.What the present invention was used is H9N2 type avian influenza virus.
The present invention produces the different of antibody in view of NS1 albumen in natural infection chicken colony with in the vaccine immune chicken colony, the average OD value of calculating chicken group serum by enzyme linked immunosorbent assay is come antidiastole.With the NS1 albumen of prokaryotic expression as antigen coated enzyme reaction plate, with the antigen-reactive of serum to be checked by certain dilution and bag quilt, the ELIAS secondary antibody colour developing that adds anti-IgG again judges by the size of measuring chicken group mean OD value whether the chicken group has infected bird flu.
The inventive method is by calculating the overall NS1 antibody horizontal of chicken group, can differentiate the chicken group and the inactivated vaccine vaccinated flock of bird flu natural infection, and the method for traditional serodiagnosis bird flu has blood clotting and hemagglutination-inhibition test, agar immunodiffusion, microneutralization and neuraminidase inhibition test, because the antibody that these methods detected, no matter be that vaccine inoculation or the wild poison of infection all can produce, therefore these detection methods can not truly reflect the actual infection conditions of bird flu, bring great difficulty to prevention and control of fowl influenza.The inventive method has been ignored individual difference from overall antidiastole, by calculating the method for the average OD value of serum, comes the whole chicken group of antidiastole whether to infect avian influenza virus from population level.In 95 parts of vaccine inoculation group serum, OD
450＞0.5 sample size accounts for 33.6%, but these chickens do not infect avian influenza virus, just causes so but vaccine valence is high.If detect from individual level, these chickens can be judged as the positive; In 70 parts of bird flu H9N2 positive serums, OD
450＞0.5 sample size accounts for 74.3%, and remaining 25.7% chicken can be judged as feminine gender from the individual level detection, and utilizes method of the present invention can differentiate whether chicken group's integral body has infected avian influenza virus, for diagnosis, birds flu-preventing provide foundation.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, for example Sambrook etc. is in the condition described in " molecular cloning laboratory manual " (New York publishing house of cold spring harbor laboratory, 1989), or the condition of advising according to manufacturer.
Present embodiment at first carries out H9N2 type avian influenza virus the inverse transcription polymerase chain reaction amplification, obtain the purpose segment, again the purpose segment is connected with carrier pET-32a (+) plasmid, obtain recombinant expression plasmid, and the importing e. coli bl21 is expressed, obtain the fusion that molecular weight is 44.2KD, this fusion obtains purity higher H 9N2 type avian influenza virus NS1 albumen through purifying; With this NS1 albumen as antigen coated enzyme reaction plate, (the best bag that specifically comprises antigen is by concentration to determine the optimum reaction condition of enzyme linked immunosorbent assay, the optimum diluting multiple of antibody, the antigen-antibody optimum reacting time, the optimum diluting multiple of ELIAS secondary antibody and the optimum reacting time of ELIAS secondary antibody); Set up the method for enzyme linked immunosorbent assay by above-mentioned optimum reaction condition, measure the enzyme linked immunosorbent assay mean light absorbency that 70 parts of bird flu H9N2 infect the negative serum of serum, 95 parts of inactivated vaccine inoculation serum, 30 parts of non-immune chickens, determine critical value, the minimum sample number that serum sample enzyme linked immunosorbent assay yin and yang attribute is judged by statistical analysis; At last according to the enzyme linked immunosorbent assay mean light absorbency of chicken group sample, from population level antidiastole chicken group whether by avian flu virus infection.
The prokaryotic expression of H9N2 type avian influenza virus NS1 albumen
The clone of H9N2 type bird flu NS1 gene
With Trizol reagent (Invitrogen company) extracted total RNA.Carry out the inverse transcription polymerase chain reaction amplification according to bird flu NS1 gene conserved sequence design primer, be connected to pMD-18 T carrier (Takara company) after the amplification and go up order-checking, comprise the primer that EcoR I restriction enzyme site and downstream comprise Xho I restriction enzyme site according to sequencing result design upstream, carry out polymerase chain reaction (PCR) amplification.
The structure of NS1 protein expression vector
With EcoR I and Xho I respectively enzyme cut above-mentioned PCR product and pET-32a (+), 10-16 ℃ of connection of spending the night of T4 dna ligase (Takara company), connecting product changes in the bacillus coli DH 5 alpha, 37 ℃ of incubated overnight, extract plasmid, cut evaluation with EcoR I and Xho I enzyme, the recombinant plasmid that evaluation is positive checks order, the result shows that the NS1 albumen homology of institute's extension increasing sequence and avian influenza virus Hong Kong strain (A/aquatic bird/HongKong/399/99) reaches 98%, so through behind the inverse transcription polymerase chain reaction, the sequence that is increased is a bird flu NS1 albumen.
The expression of NS1 albumen
Positive recombinant plasmid that will be through identifying changes in the e. coli bl21, and 1 colony inoculation 2ml of picking ampicillin LB nutrient culture media incubated overnight is got 200 μ l incubated overnight bacterium liquid inoculation 20ml ampicillin LB nutrient culture media, and 37 ℃ 150 rev/mins are shaken bacterium 2h, to OD
600Be 0.4-1, adding isopropyl sulfydryl galactoside to final concentration is 1mM, 25 ℃ shake bacterium 4h after, collect bacterium liquid.
The detection of NS1 albumen
(1) polyacrylamide gel electrophoresis
The separation gel of preparation 14% and 5% concentrated glue.Sample boils 4min with sample-loading buffer boiling water, concentrate glue voltage 80V, separation gel voltage 136V, electrophoresis 4-5h, behind the polyacrylamide gel electrophoresis, with Coomassie brilliant blue to the polyacrylamide gel 2h that dyes, decolour with destainer, observe the protein band on the gel, have tangible band to occur at the 44KD place
(2) protein immunoblotting experimental identification
When polyacrylamide gel electrophoresis finishes, keep above-mentioned polyacrylamide gel, with distilled water drip washing graphite cake, the cleaning that keeps graphite cake, wipe dried then with paper, cut 6 filter paper and a nitrocellulose filter, nitrocellulose filter is immersed on the water surface of deionized water, filter paper is immersed in the transfering buffering liquid, balance 5min, then filter paper, nitrocellulose filter and polyacrylamide gel are put from the anode to the negative electrode correctly by the order of filter paper (three), nitrocellulose filter, polyacrylamide gel, filter paper (three), according to 0.65mA/cm
2Energising transfer printing 5h, nitrocellulose filter is placed in the polybag of sealing, in add confining liquid and bird flu positive serum (the bird flu positive serum is dilution in 1: 100), 4 ℃ of night incubation, change 37 ℃ again over to and hatch 2h, after the reaction, take out nitrocellulose filter earlier with phosphate buffer (PBS) rinsing three times, each 10min, use hybond membrane cleaning fluid (TBST) rinsing 10min again, then nitrocellulose filter be placed in the polybag of sealing, in add confining liquid and 1: 1000 dilution exempt from anti-chicken IgG two anti-(Bethyl laboratories.Inc), hatch 2h for 37 ℃, after reaction finishes, nitrocellulose filter is put in the hybond membrane cleaning fluid rinsing 3 times, each 1h, add substrate colour developing 2-3min then, found that on the nitrocellulose filter has black stripe to occur at the 44KD place.
The foundation of enzyme-linked immunologic adsorption test method
Utilize the recombinant fowl influenza NS1 albumen of purifying to set up enzyme linked immunosorbent assay as antigen, the best bag of measuring antigen is by the best dilute concentration of concentration, antibody, the antigen-antibody optimum reacting time, the optimum diluting multiple of ELIAS secondary antibody, the optimum reacting time of ELIAS secondary antibody.Antigen is by 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL concentration dilutions, bag is by reaction plate 100 μ L/ holes, 4 ℃ are spent the night, and then with enzyme linked immunosorbent assay washing lotion washing 5 times, seal 2 hours for 37 ℃ with 5% skimmed milk confining liquid again; Avian flu virus infection serum, inactivated vaccine inoculation serum and negative serum are respectively with 50,100,200,400 times of antibody diluent dilutions, form square formation 100 μ L/ holes with the different dilutabilitys of above-mentioned antigen, set up the enzyme linked immunosorbent assay that detects NS1 antibody according to a conventional method, identical dilutability is done a repetition, on microplate reader, read OD
450, the OD of three kinds of serum of comparison
450Value, to select negative serum OD value be below 0.3 and the greatest dilution of the envelope antigen that virus infections and vaccine inoculation group serum OD value ratio are higher is antigen coated optium concentration, the greatest dilution of corresponding serum is best serum dilution, thereby the best bag of having determined antigen is by the suitableeest extension rate of concentration and antibody.The best bag of antigen is 40 μ g/mL by concentration, and the optimum diluting multiple of antibody is 1: 200.
Behind the best multiple diluting reaction of recombinant antigen and the H9N2 type bird flu standard positive serum 1h, with the HRP mark exempt from anti-chicken igg antibody by dilution in 1: 500,1: 1000,1: 10000,1: 100000, reaction 1h adds substrate solution chromogenic assay OD again
450, when OD occurring
450Two maximum anti-concentration are best dilute concentration.The optimum diluting multiple of ELIAS secondary antibody is 1: 10000.
Each 6 parts of recombinant antigen and H9N2 type bird flu standard positive serum, negative serums react 0.5h, 1h, 1.5h respectively by after the best multiple dilution, add ELIAS secondary antibody, and OD is measured in colour developing
450, the reaction time when positive and negative serum ratio maximum is optimum reacting time.The antigen-antibody optimum reacting time is 1h.
After antigen and 4 parts of H9N2 type bird flu standard positive serums, negative serums diluted by best multiple, the reaction certain hour added ELIAS secondary antibody, reacts 0.5h, 1h, 1.5h respectively, chromogenic assay OD
450, the reaction time when positive and negative serum ratio maximum is the optimum reacting time of ELIAS secondary antibody.The ELIAS secondary antibody optimum reacting time is 1h.
The formulation of enzyme linked immunosorbent assay yin and yang attribute critical value and minimum sample number
By measuring the OD value that 70 parts of bird flu H9N2 infect the negative serum of serum, 95 parts of inactivated vaccine inoculation serum, 30 parts of non-immune chickens, vaccine inoculation group sero-enzyme linked immunosorbent adsorption test OD
450The mean value X=0.428 of value, standard deviation SD=0.228.Bird flu H9N2 infects serum group enzyme linked immunosorbent assay OD
450The mean value X=0.576 of value, standard deviation SD=0.151.Reference literature (" veterinary epidemiology principle ", Liu Xiu chief editor of ancient India, agriculture publishing house) carries out t check, t=5.016 to the two.T＞t
83,0.05 (bilaterals)(83 degree of freedom) for the t check, significant difference.Promptly can be at population level with inactivated vaccine inoculation group serum and the difference of bird flu H9N2 infected group serum by enzyme linked immunosorbent assay.
The criterion that present embodiment adopts is that the mean value of negative sample adds three standard deviations, and the mean value of non-immune chicken negative serum group is X=0.222, standard deviation SD=0.09221.Judge yin and yang attribute result's OD
450Critical value is X+3SD=0.5, if the OD of colony's serum
450Mean value is higher than 0.5, then is judged to be the positive; If be lower than 0.5, be judged to be feminine gender.
Present embodiment is to detect the chicken group from integral level whether to infect avian influenza virus.By check chicken group mean OD
450Judge whether the chicken group all infects avian influenza virus.The regulation of experiment sample number in the list of references (" veterinary epidemiology principle ", Liu Xiu chief editor of ancient India, agriculture publishing house, 147-148 page or leaf):
P is OD in the infection group and viral infection group sample
450The percentage that＞0.5 sample size is shared; Q is OD in the vaccine immunity group sample
450The percentage that＞0.5 sample size is shared; N is required animal used as test number.
In 95 parts of vaccine inoculation group serum, OD
450＞0.5 sample size accounts for 33.6%; In 70 parts of bird flu H9N2 positive serums, OD
450＞0.5 sample size accounts for 74.3%.According to above formula, obtaining testing the serum sample number is 30 parts.So when carrying out colony's detection, the experiment serum sample number that is detected is at least 30, can differentiate effectively just whether the chicken group infects avian influenza virus.
Enzyme linked immunosorbent assay antidiastole avian influenza virus natural infection chicken group
After the NS1 albumen of purifying was diluted to 40 μ g/ml with the diluted liquid of bag, every hole added 100 μ l, and 4 ℃ are spent the night, and behind 37 ℃ of 2h, discard the liquid hole in, select 37 ℃ of sealings of 5% skimmed milk 2h again, discard liquid in the hole then, lavation buffer solution (PBST) cleaning 5 times; With 1: 200 times of dilution of test serum sample, every hole adds 100 μ l, and 37 ℃ of 1h discard liquid in the hole, cleans 5 times with lavation buffer solution, adds enzyme thereafter and marks anti-IgG antibody 100 μ l, and 37 ℃ of 1h discard liquid in the hole, cleans 5 times with the lavation buffer solution washing lotion.Add 3,3 ', 5 again, 5 '-tetramethyl benzidine (TMD), 100 μ l color development at room temperature 8min add 50 μ l stop buffer cessation reactions thereafter, measure OD
450Result of determination.When the quantity of the chicken that is detected greater than 30 the time, the average OD of the chicken serum that detects
450Greater than 0.5 o'clock, judge that the chicken group has infected bird flu, as the average OD of detection chicken serum
450Less than 0.5 o'clock, judge that the chicken group does not infect bird flu.
Claims (8)
1, the chicken infected enzyme-linked immunologic adsorption test method of a kind of antidiastole bird flu is characterized in that, described method concrete steps comprise:
1. the gene by inverse transcription polymerase chain reaction amplification H9N2 type avian influenza virus NS1 albumen;
2. the gene of gained H9N2 type avian influenza virus NS1 albumen of 1. step being increased is cut by enzyme and is inserted among the vector plasmid pET-32a (+), construct prokaryotic expression plasmid, the plasmid that builds is changed in the e. coli bl21, isopropyl sulfydryl galactoside abduction delivering, expression product is a fusion through polyacrylamide gel electrophoresis and protein immunoblotting experimental identification;
3. with step 2. the gained fusion set up the method for enzyme linked immunosorbent assay as antigen coated enzyme reaction plate, determine reaction conditions;
4. the method for the enzyme linked immunosorbent assay of 3. setting up according to step detects negative serum, the bird flu H9N2 inactivated vaccine inoculation group chicken serum of bird flu H9N2 infected group chicken serum, the non-immune chicken of 30 ages in days;
5. according to statistical method testing result is analyzed, determined the critical value and the minimum sample number of the enzyme linked immunosorbent assay yin and yang attribute judgement of bird flu serum;
6. according to the critical value of serum sample enzyme linked immunosorbent assay yin and yang attribute judgement and the minimum detection sample number of regulation, according to the size of the enzyme linked immunosorbent assay mean light absorbency of chicken group serum sample, the chicken group that is defined as that mean light absorbency is higher than critical value has infected the wild poison of bird flu.
2, the chicken infected enzyme-linked immunologic adsorption test method of antidiastole bird flu according to claim 1 is characterized in that, the fusion of step described in 2., and its molecular weight is 44.2KD.
3, the chicken infected enzyme-linked immunologic adsorption test method of antidiastole bird flu according to claim 1, it is characterized in that, the enzyme linked immunosorbent assay of step described in 3., its reaction conditions is: the bag of antigen is 40 μ g/mL by concentration, the extension rate of antibody is 1: 200, the antigen-antibody reaction time is 1h, and the extension rate of ELIAS secondary antibody is 1: 10000, and the reaction time of ELIAS secondary antibody is 1h.
4, the chicken infected enzyme-linked immunologic adsorption test method of antidiastole bird flu according to claim 1, it is characterized in that, the critical value that the bird flu sero-enzyme linked immunosorbent adsorption test yin and yang attribute of step described in 4. judged, be by after the negative serum of 70 parts of bird flu H9N2 infected group chicken serums, 30 part of 30 non-immune chicken of age in days, 95 parts of bird flu H9N2 inactivated vaccine inoculation group chicken serums are carried out statistical analysis, the mean value of getting the negative serum of the non-immune chicken of 30 ages in days adds three standard deviations and gets.
5, the chicken infected enzyme-linked immunologic adsorption test method of antidiastole bird flu according to claim 4 is characterized in that, the critical value that the bird flu sero-enzyme linked immunosorbent adsorption test yin and yang attribute of step described in 4. judged is 0.5.
6, the chicken infected enzyme-linked immunologic adsorption test method of antidiastole bird flu according to claim 1, it is characterized in that, the minimum detection sample number of step described in 4. is by statistical analysis, and the regulation enzyme linked immunosorbent assay determines whether chicken group integral body infects the number of the needed minimum chicken of avian influenza virus.
7, the chicken infected enzyme-linked immunologic adsorption test method of antidiastole bird flu according to claim 6 is characterized in that, the minimum detection sample number of step described in 4. is 30.
8, the chicken infected enzyme-linked immunologic adsorption test method of antidiastole bird flu according to claim 1, it is characterized in that, the size according to the enzyme linked immunosorbent assay mean light absorbency of chicken group serum sample of step described in 5., determine whether the chicken group infects the wild poison of bird flu, be meant: the enzyme linked immunosorbent assay mean light absorbency of chicken group serum sample is greater than 0.5, and the chicken group infects avian influenza virus; The enzyme linked immunosorbent assay mean light absorbency of chicken group serum sample is less than 0.5, and the chicken group does not infect the wild poison of bird flu.
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Cited By (3)
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| CN103901213A (en) * | 2014-03-31 | 2014-07-02 | 山东农业大学 | Detection method for distinguishing generation of antibody through activated vaccine and inactivated vaccine of tembusu virus |
| CN104749372A (en) * | 2013-12-30 | 2015-07-01 | 北京义翘神州生物技术有限公司 | ELISA kit for H9N2 influenza virus hemagglutinin protein |
| CN114230642A (en) * | 2021-10-28 | 2022-03-25 | 扬州大学 | Polypeptide for distinguishing H7N9 subtype avian influenza virus infection from immune animals and detection method |
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2006
- 2006-12-28 CN CN 200610148212 patent/CN1996021A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104749372A (en) * | 2013-12-30 | 2015-07-01 | 北京义翘神州生物技术有限公司 | ELISA kit for H9N2 influenza virus hemagglutinin protein |
| CN103901213A (en) * | 2014-03-31 | 2014-07-02 | 山东农业大学 | Detection method for distinguishing generation of antibody through activated vaccine and inactivated vaccine of tembusu virus |
| CN103901213B (en) * | 2014-03-31 | 2016-04-13 | 山东农业大学 | A kind of tembusu virus poison of living of distinguishing produces the detection method of antibody with inactivated vaccine |
| CN114230642A (en) * | 2021-10-28 | 2022-03-25 | 扬州大学 | Polypeptide for distinguishing H7N9 subtype avian influenza virus infection from immune animals and detection method |
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