CN106442986B - A kind of method of the detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose - Google Patents
A kind of method of the detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose Download PDFInfo
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Abstract
The present invention relates to a kind of methods of detection zone swine influenza virus subtype distribution, belong to animal health detection technique field, which is characterized in that comprise the following steps：Blood serum sample is gathered first, blood serum sample is handled using " pancreatin Na2Fe04 hydrogen peroxide polyethylene glycol " method, is removed non-specific clotting factor therein, is obtained serum to be checked；The antigen concentration of 4 blood coagulation units is determined using the method for doubling dilution；Serum to be checked, antigen to be checked and chicken erythrocyte suspension is made to react on V-type micro-reaction plate, using the highest extension rate of the serum to be checked of 4 blood coagulation unit antigen to be checked of complete inhibition as the potency of serum antibody, determine serum antibody titer for positive serum to be checked.The method of the invention is simple and effective, at low cost, and positive rate analysis is carried out available for a fairly large number of blood serum sample, lays the foundation for Animal diseases treatment with prevention.
Description
Technical field
The invention belongs to animal health detection technique field, more particularly to a kind of inspection of non-disease diagnose and treat purpose
The method for surveying regional swine influenza virus subtype distribution.
Background technology
Swine influenza virus (Swine Influenza Virus, SIV) belongs to orthomyxoviridae family, A types influenza disease in classification
Poison belongs to, and sub-thread minus-stranded rna virus, genome is made of 8 independent segments to differ in size, can cause the three classes animal of a boar
Epidemic disease, H1N1, H3N2 and H1N2 hypotype swine influenza virus are the strains for being widely present and frequently causing swinery to fall ill in swinery.It is single
Pure SIV infection has the characteristics that incidence is high, the death rate is low, but swine flu is typical inhibitive ability of immunity disease, can be caused
The mixing or scabies secondary infection of other cause of diseases, in turn result in serious economic loss.
After the big influenzas of Spain in 1918, influenza infection swinery develops into classical swine flu (H1/CS) branch,
Europe H1N1 hypotype fowl source streams Influenza Virus infection swinery in 1976, and CS branches are gradually substituted, become swine influenza virus and mainly divide
Branch, i.e., Eurasian class fowl pig source stream Influenza Virus branch (H1/EA), the influenza disease that Hong-Kong was separated to EA branches for the first time in 2001
Poison, in addition H1 and H3 hypotypes seasonal influenza (HS) apparent regionality distribution is then presented in swinery.2009 from Mexico
Broken out the flu outbreak have swept the globe, and the wide-scale distribution in Chinese swinery, by show after phylogenetic analysis pig source,
It is exactly the pdm/09 newly broken out that people source and fowl source stream Influenza Virus, which recombinate the novel influenza to be formed,.
The airway epithelial cell of pig has while express alpha -2,3 and α -2, the ability of the receptor of 6 two kinds of influenza viruses, because
And pig can infect people, pig and fowl source stream Influenza Virus simultaneously, it is influenza virus " mixer " to generally believe pig.It is in Chinese swinery
The present situation that existing a variety of subtype influenza virus coexist, from after pdm/09 infection of Chinese swinerys, pdm/09 is increasingly becoming in Chinese swinery
Main prevalence branch, and start to be widely present in Chinese swinery.Research shows that pdm/09 is sent out with other influenza viruses
Raw restructuring forms novel influenza, and starts to propagate in swinery.So the detection for strengthening swinery influenza virus is defended to public
Life has very important significance, and domestic and foreign scholars have done numerous studies to the diagnosis of swine influenza virus with detection therefore, develop
Go out the detection methods such as PCR, ELISA and Real-Time PCR, but these methods it is bothersome laborious, it is necessary to expensive laboratory apparatus and
Skilled operative skill is completed, and is not suitable for the distribution situation of detection zone swine influenza virus, common is also most basic
The method of detection zone swine influenza virus distribution situation be blood clotting and hemagglutination-inhibition test, but due to containing in animal blood serum
Non-specific clotting factor often results in the false positive of result, to detection work cause to hinder, additionally, due to detection heavy workload,
Test sample is more, in the analysis of result data, easily causes disorder.
The content of the invention
In order to solve the problems, such as that blood clotting and hemagglutination-inhibition test are disturbed by non-specific clotting factor and data analysis is difficult,
The present invention intends providing a kind of method of the detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose, the party
Method is simple and effective, of low cost, suitable for the development of region detection work.
The method of the detection zone swine influenza virus subtype distribution of a kind of non-disease diagnose and treat purpose, including following
Step：
Step 1: acquisition blood serum sample, is divided into different groups, to every part of blood serum sample in every group according to different location
It is handled, processing step is：The trypsin solution of 15 μ L 8mg/mL, 56 DEG C of water bath processings are added in every 30 μ L blood serum samples
30min adds in Na2Fe04 aqueous solution and the 60 μ L percentage by volumes of 60 μ L 2.3mg/mL as 3% aqueous hydrogen peroxide solution,
15min is stored at room temperature after mixing, then adds the Aqueous Solutions of Polyethylene Glycol that 60 μ L mass percentage by volumes are 0.5%, room temperature after mixing
10min is stood, is mixed with the PBS solution of 75 μ L, obtains serum to be checked；Take the chicken red blood cell that serum to be checked and volume fraction are 1%
Suspension, according to 1:1 volume ratio mixes the two, and chicken red blood cell sinks in teardrop shaped, and blood serum sample is handled successfully, to be checked
Serum is used for subsequent step；
Step 2: the PBS solution of 50 μ L is separately added into the 2-12 holes of V-type micro-reaction plate；It is added in the 1st hole
100 μ L antigens to be checked, are suctioned out 50 μ L and are placed in the 2nd hole, and suctioning out 50 μ L from the 2nd hole after mixing is placed in the 3rd hole, according to
It is secondary to carry out serial doubling dilution to the 11st hole, abandon 50 final μ L；1% chicken red blood cell of 50 μ L is added in the 1st to 12 hole
V-type micro-reaction plate is placed on micro oscillator and handles 1-2min by suspension, and 37 DEG C of standing 15min, observation is treated as a result, obtaining
Examine the blood coagulation unit of antigen；
Step 3: it is the antigen to be checked of 4 blood coagulation units according to the result compound concentration of step 2, in V-type micro-reaction
The PBS solution of 25 μ L is separately added into 11 holes of 2- of plate, the 12nd hole adds in the PBS solution of 50 μ L；It is added in the 1st hole at 50 μ L
The serum to be checked managed, is suctioned out 25 μ L and is placed in the 2nd hole, and suctioning out 25 μ L from the 2nd hole after mixing is placed in the 3rd hole, according to
It is secondary to carry out serial doubling dilution to the 10th hole, abandon 25 final μ L；25 μ L concentration are separately added into 1-11 holes as 4 blood
The antigen to be checked of solidifying unit, 37 DEG C stand 30 minutes, and 1% chicken erythrocyte suspension of 50 μ L is separately added into 1-12 holes；With
Completely inhibit potency of the serum highest extension rate to be checked of 4 blood coagulation unit antigen to be checked as serum antibody, serum antibody
It is the positive during potency >=40；
Step 4: statistical analysis, the difference of more different groups of seroprevalences to be checked are carried out to the data that step 3 obtains
Whether significantly.
Optimize as further, the preparation method of trypsin solution in step 1：Add in 15mL's in every 0.8g pancreatin
D-Hank ' s liquid is modulated into paste, and D-Hank ' the s liquid for adding 40mL is stirred to being completely dissolved, and is settled to 100mL, is used carbonic acid
Hydrogen sodium solution tune pH to 7.2, filtration sterilization are spare.
As further optimization, V-type micro-reaction plate is cleaned before use, and cleaning step is：It will be immersed with
The cotton swab of 75% ethyl alcohol, which is placed in every hole of V-type micro-reaction plate, to be rotated, and then with distilled water flushing 2 times, then is washed with distilled water
It washs 3 times, puts dry in 37 DEG C of incubators.
As further optimization, described 1% chicken erythrocyte suspension is by the mixing blood of 3 SPF chickens, is added to it
In the Alsever liquid of 2 times of volumes, mixing shakes up；500rpm horizontal centrifugal 5min, the Alsever liquid and blood cell for suctioning out upper strata sink
The leukocytic cream on starch surface obtains blood cell sediment；The physiological saline of the pH=7.2 of blood cell 20 times of volumes of sediment is added in, is mixed
It closes uniformly, 1500rpm centrifugations 10min is washed, and is repeated washing 3 times after suctioning out supernatant, is taken precipitation；It will precipitation and physiology salt
Water is with 1:100 volume ratio is uniformly mixed, and obtains 1% chicken erythrocyte suspension.
Advantageous effect：
The present invention acts on processing blood serum sample successively using pancreatin, Na2Fe04, hydrogen peroxide and polyethylene glycol, wherein,
Pancreatin makes iuntercellular junction protein degradation, at this moment cell is due to therein cell by protein degradation on location
Become spherical shape under the tension force effect of skeleton, so that cell separates.The action of atomic oxygen that hydrogen peroxide is discharged with Na2Fe04 is not
Stablize non-specific castle's intrinsic factor, it is made to tend towards stability, and Na2Fe04 itself is reduced, and has certain flocculating effect, can be incited somebody to action
Remaining leucocyte fragment and the non-specific castle's intrinsic factor wadding collection stablized, using the inducing action of polyethylene glycol, sink in serum
Shallow lake macromolecular substances eliminate the influence to erythrocyte agglutination, while polyethylene glycol makes all kinds of substances in serum be in evacuation and live
Jump state is that the reaction with chicken red blood cell creates good environment, and the method is simple and effective, convenient and efficient, with commonly using non-spy
The minimizing technology of different in nature agglutination factor is compared, with obvious effects, and aggegation rate is reduced to 0, eliminates the interference of the nonspecific agglutination factor.
Specific embodiment
Below by specific embodiment, the present invention will be further explained.
The method of the detection zone swine influenza virus subtype distribution of a kind of non-disease diagnose and treat purpose, including following
Step：
Step 1: acquisition blood serum sample, is divided into different groups, to every part of blood serum sample in every group according to different location
It is handled, processing step is：The trypsin solution of 15 μ L 8mg/mL, 56 DEG C of water bath processings are added in every 30 μ L blood serum samples
30min adds in Na2Fe04 aqueous solution and the 60 μ L percentage by volumes of 60 μ L 2.3mg/mL as 3% aqueous hydrogen peroxide solution,
15min is stored at room temperature after mixing, then adds the Aqueous Solutions of Polyethylene Glycol that 60 μ L mass percentage by volumes are 0.5%, room temperature after mixing
10min is stood, is mixed with the phosphate buffer (i.e. PBS solution) of 75 μ L, obtains serum to be checked；Take serum to be checked and volume fraction
For 1% chicken erythrocyte suspension, according to 1:1 volume ratio mixes the two, and chicken red blood cell sinks in teardrop shaped, serum sample
Product are handled successfully, and serum to be checked is used for subsequent step；
Wherein, the preparation method of trypsin solution：Balanced salt solution (i.e. D-Hank ' the s of 15mL are added in every 0.8g pancreatin
Liquid) paste is modulated into, D-Hank ' the s liquid for adding 40mL is stirred to being completely dissolved, and is settled to 100mL, is used sodium bicarbonate solution
PH to 7.2 is adjusted, filtration sterilization is spare.
Wherein, 1% chicken erythrocyte suspension is the Alsever's Solution that the mixing blood of 3 SPF chickens is added to its 2 times of volumes
In (i.e. Alsever liquid), mixing shakes up；500rpm horizontal centrifugal 5min suction out the Alsever liquid on upper strata and blood cell sediment table
The leukocytic cream in face obtains blood cell sediment；The physiological saline of the pH=7.2 of blood cell 20 times of volumes of sediment is added in, is uniformly mixed,
1500rpm centrifugations 10min is washed, and is repeated washing 3 times after suctioning out supernatant, is taken precipitation；It will precipitate with physiological saline with 1:
100 volume ratio is uniformly mixed, and obtains 1% chicken erythrocyte suspension.
Step 2: the PBS solution of 50 μ L is separately added into the 2-12 holes of V-type micro-reaction plate；It is added in the 1st hole
100 μ L antigens to be checked, are suctioned out 50 μ L and are placed in the 2nd hole, and suctioning out 50 μ L from the 2nd hole after mixing is placed in the 3rd hole, according to
It is secondary to carry out serial doubling dilution to the 11st hole, abandon 50 final μ L；1% chicken red blood cell of 50 μ L is added in the 1st to 12 hole
V-type micro-reaction plate is placed on micro oscillator and handles 1-2min by suspension, and 37 DEG C of standing 15min, observation is treated as a result, obtaining
Examine the blood coagulation unit of antigen；
Wherein, V-type micro-reaction plate is cleaned before use, and cleaning step is：It will be immersed with the cotton swab of 75% ethyl alcohol
It is placed in every hole of V type micro-reaction plates and rotates, then with distilled water flushing 2 times, then washed 3 times with distilled water, put 37 DEG C of temperature
It is dry in case.
Step 3: it is the antigen to be checked of 4 blood coagulation units according to the result compound concentration of step 2, in V-type micro-reaction
The PBS solution of 25 μ L is separately added into the 2-11 holes of plate, the 12nd hole adds in the PBS solution of 50 μ L；50 μ L processing are added in the 1st hole
Good serum to be checked, being suctioned out 25 μ L is placed in the 2nd hole, and suctioning out 25 μ L from the 2nd hole after mixing is placed in the 3rd hole, successively
Serial doubling dilution is carried out to the 10th hole, abandons 25 final μ L；25 μ L concentration are separately added into 1-11 holes as 4 blood clotting lists
The antigen to be checked of position, 37 DEG C stand 30 minutes, and 1% chicken erythrocyte suspension of 50 μ L is separately added into 1-12 holes；With complete
Inhibit potency of the serum highest extension rate to be checked of 4 blood coagulation unit antigen to be checked as serum antibody, serum antibody titer
It is the positive when >=40；
Step 4: carrying out statistical analysis to the data that step 3 obtains, seroprevalence to be checked during different sample sizes is calculated
95% confidence interval, whether the difference of more different groups of seroprevalences to be checked notable.
To nonspecific agglutination factor minimizing technology effect measuring：
40 blood serum samples are handled respectively with following 4 kinds of methods (A, B, C, D), with 1% chicken erythrocyte suspension to above-mentioned place
The blood serum sample of reason carries out hemagglutination test, measures the removal effect of nonspecific agglutination phenomenon.
A：Blood serum sample directly measures；
B：56 DEG C of water bath processing 30min of blood serum sample are measured after letting cool；
C：It is measured after the processing of " pancreatin-potassium metaperiodate-glycerine " method；
D：It is measured after being handled using the method for the present invention.
Measurement result is as shown in table 1 below.
Wherein, " 1-2 holes " represents that the number being aggregated occur in only 1-2 holes；" 3 holes and more than " represent 3 holes and appear above solidifying
The number of collection.
The result shows that the nonspecific agglutination rate of untreated blood serum sample is up to 65%, after thermally treated, non-spy
Different in nature aggegation rate decreases, and drops to 37.5%, and common " pancreatin-potassium metaperiodate-glycerine " method effect is slightly notable, but still
Hemagglutination-inhibition test can be had an impact, after being handled with heretofore described method, nonspecific agglutination phenomenon disappears, for blood
The solidifying blood serum sample for inhibiting experiment will not generate false positive.
Embodiment 1 carries out Jiangxi Province influenza virus using a kind of method of detection zone swine influenza virus subtype distribution
Serosurvey
By carrying out serological surveillance and the statistical analysis of H1 and H3 hypotype swine influenza viruses, Jin Erzhang to Jiangxi Province swinery
Hold the popularity of influenza virus in swinery in Jiangxi Province.
Step 1: the collection of sample and information
From in October, 2012 to the serum totally 2005 between in March, 2015, having collected covering scope farm of the whole province of Jiangxi Province
Part, wherein being within 2012 130 parts, 2013 are 430 parts.2014 are 924 parts, and 2015 are 548 parts.Each blood of establishing district and municipality
Clear different, wherein Fuzhou Shi and Ji'an City is most, is 562 and 338 parts, be secondly Ganzhou City, Shangrao City, Nanchang City, Jing Dezhen,
Yichun City and Jiujiang City, respectively 205,172,158,155,139 and 126 parts, wherein Pingxiang City, Xinyu City and Yingtan are most
It is few, it is respectively 65,55 and 30 parts.
Step 2: the collection and processing of blood serum sample
The serum for coming from each interior pig farm of establishing district and municipality in Jiangxi Province is collected, 15 parts of serum are at least collected by each farm.To blood
Final proof product are handled, and remove the non-specific clotting factor in serum, and in 4 DEG C of preservations.It concretely comprises the following steps：In every 30 μ L blood
The trypsin solution of 15 μ L 8mg/mL, 56 DEG C of water bath processing 30min, the ferric acid of 60 μ L 2.3mg/mL of addition are added in final proof product
Sodium water solution and 60 μ L percentage by volumes are 3% aqueous hydrogen peroxide solution, and 15min is stored at room temperature after mixing, then adds 60 μ L matter
The Aqueous Solutions of Polyethylene Glycol that percentage by volume is 0.5% is measured, 10min is stored at room temperature after mixing, mixes, obtain with 75 μ LPBS solution
Serum to be checked；Serum to be checked is taken, according to 1:1 volume ratio is mixed with 1% chicken erythrocyte suspension, and chicken red blood cell is under teardrop shaped
Heavy, blood serum sample is handled successfully, and serum to be checked is used for subsequent step.
Step 3: the detection and statistical analysis of Serum HI antibody
The blood coagulation unit of antigen is selected in detection first, and 50 μ L PBS are separately added into the 2-12 holes of V-type micro-reaction plate,
100 μ L antigens to be checked are added in the 1st hole, 50 μ L is suctioned out and is placed in the 2nd hole, successively according to 1/2 times of doubling dilution to the 11st
50 final μ L are abandoned in hole, and 1% chicken erythrocyte suspension of 50 μ L is added in the 1st to 12 hole, and 37 DEG C stand 15 minutes, observation
As a result, obtain the blood coagulation unit with reference to antigen.
Result judgement：As a result with ++++, +++, ++ ,+, ± ,-represent, wherein：
One layer of red blood cell, which is equably laid on hole wall, is ++++；
One layer of red blood cell is uniformly laid on hole wall, but edge is irregular, and the area of paving is slightly smaller to be +++；
Red blood cell formed a ring-type, surrounding have it is small aggegation block be ++；
Red blood cell forms a small group, but edge is rough, surrounding have small grumeleuse for ±；
Red blood cell formed a small circle, edge without any small grumeleuse for-.
With ++ for terminal, i.e., the inverse of the highest dilution of erythrocyte agglutination can be completely inhibited as terminal.Obtain a blood clotting
Titre.Hemagglutination titer divided by 4 are obtained into extension rate, canonical reference antigen by this multiple is diluted, is 4 blood coagulation units.
Compound concentration is the antigen to be checked of 4 blood coagulation units, and 25 μ are separately added into the 2-11 holes of V-type micro-reaction plate
L PBS, the 12nd hole add in 50 μ L PBS, and the serum to be checked that 50 μ L are handled well is added in the 1st hole, suction out 25 μ L and are placed in the 2nd hole,
Successively according to 1/2 times of doubling dilution to the 10th hole, 25 final μ L are abandoned；25 μ L concentration are separately added into 1-11 holes as 4
The antigen to be checked of blood coagulation unit, 37 DEG C stand 30 minutes, and 1% chicken erythrocyte suspension of 50 μ L is separately added into 1-12 holes；
To completely inhibit the serum highest extension rate to be checked of 4 blood coagulation unit antigen to be checked as the potency of serum antibody, serum resists
It is the positive during body potency >=40.
Step 4: data statistics and analysis
Statistical analysis is carried out to the data of acquisition using IBM SPSS Statistics 20 and Graphpad Prism 5,
Whether 95% confidence interval of seroprevalence when calculating different sample sizes, the difference of more different groups of seroprevalences show
It writes.As a result it is as follows：
(1) H1 and H3 subtype influenza virus popularity
The antibody level of influenza virus in the 2005 parts of serum collected in Jiangxi Province is had detected by hemagglutination-inhibition test, wherein
H1 total positiveses rate is 34.76%, and 95% confidence interval is 32.68%-36.89%, is significantly higher than other subtype influenza virus, H3
Influenza virus positive rate of antibody is 23.69%.The serum antibody of pdm/09 branches influenza virus in H1 subtype influenza virus
Positive rate highest reaches 28.73%, and is significantly higher than the influenza virus of other branches, is followed successively by H1/CS and H1/EA thereafter.Knot
Fruit is as shown in table 2 below.
(2) H1 and H3 subtype influenza virus coinfection situation
If hemagglutination-inhibition test detect antibody positive, then it is assumed that once infected the influenza virus of this branch, if two kinds or
The antibody of antigen more than person occurs positive, then it is assumed that and this pig once infected the influenza virus of two even more than two branches,
By carrying out statistical analysis discovery to experimental data：There are substantial amounts of coinfection case, pdm/ between multiple branches of H1 hypotypes
The coinfection rate that coinfection rate between 09 and EA/H1N1 is 11.62%, pdm/09 and CS is 6.38%, and EA and CS is total to
Infection rate is 5.19%；H1 and H3 subtype influenza virus antibody positive rate also goes out for the influenza virus of each branches of 6.78%, H1
Now with the coinfection case of H3 subtype influenza virus.As a result it is as shown in table 3 below.
(3) popularity of Jiangxi Province different regions influenza virus
The serological testing result of the H1 and H3 subtype influenza virus of establishing district and municipality to 11 in Jiangxi Province carries out statistical
After analysis, the antibody positive rate highest of the influenza virus of the pdm/09 branches of Shangrao City and Ji'an City is found, be respectively 44.77% He
29.88%, and it is significantly higher than the antibody positive rate of other branch influenza viruses of this area；Yingtan, Jindezhen City, Xinyu City
It is respectively 50.00%, 25.16%, 43.64% and 26.62% with the antibody positive rate highest of the H3/L22 of Yichun City, and
It is significantly higher than the antibody positive rate of the SIV of other local branches；The antibody of the pdm/09 and H1/CS branches influenza virus of Nanchang City
Positive rate highest, be respectively 49.37% and 41.77%, Liang Ge branch influenza virus antibody positive rate difference it is not notable；Jiujiang
The Antibody of Influenza positive rate highest of the pdm/09 and H3/L22 branches of swinery in city, Ganzhou City, Pingxiang City and Fuzhou Shi, and
It is significantly higher than the positive rate of H1/EA and H1/CS.As a result it is as shown in table 4 below.
(4) 2012 years to 2015 Jiangxi Province's swinery influenza virus popularities
After carrying out statistical analysis to 2012 to 2015 different branches influenza virus seroprevalences, 2013 are found
The positive rate of antibody of pdm/09 branches influenza virus is 36.74% in the serum of year Jiangxi Province's collection, is significantly higher than other
Time, and it is significantly higher than other branches of the same period and H3 subtype influenza virus antibody positive rates, it begins to decline within 2014, by 2015
Positive rate is reduced to 20.44%；With pdm/09 on the contrary, the seroprevalence of H3 influenza viruses is gradually increasing, by 2012
4.85% rises to 41.24% in 2015.The antibody positive rate of H1/HS is stablized relatively between 2012 to 2015, and H1/
CS and H1/EA then reached highest in 2014, and positive rate respectively reaches 17.21% and 16.23%.As a result such as table 3 and the following table 5 institute
Show.
Claims (5)
1. a kind of method of the detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose, feature exist
In comprising the following steps：
Step 1: acquisition blood serum sample, different groups are divided into according to different location, every part of blood serum sample in every group is carried out
Processing, processing step are：The trypsin solution of 15 μ L 8mg/mL, 56 DEG C of water bath processings are added in every 30 μ L blood serum samples
30min adds in Na2Fe04 aqueous solution and the 60 μ L percentage by volumes of 60 μ L 2.3mg/mL as 3% aqueous hydrogen peroxide solution,
15min is stored at room temperature after mixing, then adds the Aqueous Solutions of Polyethylene Glycol that 60 μ L mass percentage by volumes are 0.5%, room temperature is quiet after mixing
10min is put, is mixed with the PBS solution of 75 μ L, obtains serum to be checked；Serum to be checked and volume fraction is taken to be hanged for 1% chicken red blood cell
Liquid, according to 1:1 volume ratio mixes the two, and chicken red blood cell sinks in teardrop shaped, and blood serum sample is handled successfully, blood to be checked
Subsequent step is used for clearly；
Step 2: the PBS solution of 50 μ L is separately added into the 2-12 holes of V-type micro-reaction plate；100 μ L are added in the 1st hole
Antigen to be checked, is suctioned out 50 μ L and is placed in the 2nd hole, and suctioning out 50 μ L from the 2nd hole after mixing is placed in the 3rd hole, carries out successively
Serial doubling dilution abandons 50 final μ L to the 11st hole；1% chicken erythrocyte suspension of 50 μ L is added in the 1st to 12 hole, by V
Type micro-reaction plate, which is placed on micro oscillator, handles 1-2min, and 37 DEG C of standing 15min are observed as a result, obtaining antigen to be checked
Blood coagulation unit；
Step 3: it is the antigen to be checked of 4 blood coagulation units according to the result compound concentration of step 2, in V-type micro-reaction plate
The PBS solution of 25 μ L is separately added into 2-11 holes, the 12nd hole adds in the PBS solution of 50 μ L；Add in what 50 μ L were handled well in the 1st hole
Serum to be checked, is suctioned out 25 μ L and is placed in the 2nd hole, and suctioning out 25 μ L from the 2nd hole after mixing is placed in the 3rd hole, carries out successively
Serial doubling dilution abandons 25 final μ L to the 10th hole；25 μ L concentration are separately added into 1-11 holes as 4 blood coagulation units
Antigen to be checked, 37 DEG C stand 30 minutes, and 1% chicken erythrocyte suspension of 50 μ L is separately added into 1-12 holes；With complete inhibition
Potency of the serum highest extension rate to be checked of 4 blood coagulation unit antigen to be checked as serum antibody, serum antibody titer >=40
When for the positive；
Step 4: carrying out statistical analysis to the data that step 3 obtains, whether the difference of more different groups of seroprevalences to be checked
Significantly.
2. a kind of detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose as described in claim 1
Method, it is characterised in that：The preparation method of trypsin solution in step 1：D-Hank ' the s of 15mL are added in every 0.8g pancreatin
Liquid is modulated into paste, and D-Hank ' the s liquid for adding 40mL is stirred to being completely dissolved, and is settled to 100mL, is used sodium bicarbonate solution
PH to 7.2 is adjusted, filtration sterilization is spare.
3. a kind of detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose as described in claim 1
Method, it is characterised in that：V-type micro-reaction plate is cleaned before use.
4. a kind of detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose as described in claim 1
Method, it is characterised in that：The cleaning step of V-type micro-reaction plate is：It is micro by V-type is placed in immersed with the cotton swab of 75% ethyl alcohol
It is rotated in every hole of reaction plate, then with distilled water flushing 2 times, then wash 3 times with distilled water, put in 37 DEG C of incubators and dry.
5. a kind of detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose as described in claim 1
Method, it is characterised in that：Described 1% chicken erythrocyte suspension is by the mixing blood of 3 SPF chickens, is added to its 2 times of volumes
Alsever liquid in, mixing shakes up；500rpm horizontal centrifugal 5min suction out the Alsever liquid on upper strata and blood cell sediment surface
Leukocytic cream, obtain blood cell sediment；The physiological saline of pH=7.2 of blood cell 20 times of volumes of sediment is added in, is uniformly mixed,
1500rpm centrifugations 10min is washed, and is repeated washing 3 times after suctioning out supernatant, is taken precipitation；It will precipitate with physiological saline with 1:
100 volume ratio is uniformly mixed, and obtains 1% chicken erythrocyte suspension.
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