CN106442986A - Method for detecting regional SIV (swine influenza virus) subtype distribution - Google Patents

Abstract

The invention relates to a method for detecting the distribution of regional swine influenza virus subtypes, which belongs to the technical field of animal health detection, and is characterized in that it comprises the following steps: first collecting serum samples, using "pancreatin-sodium ferrate-hydrogen peroxide- Polyethylene glycol" method was used to process serum samples to remove the non-specific coagulation factors to obtain the serum to be tested; the method of doubling dilution was used to determine the antigen concentration of 4 hemagglutination units; the serum to be tested was prepared on a V-type microplate 1. The antigen to be tested reacts with the chicken red blood cell suspension, and the highest dilution factor of the serum to be tested that completely inhibits the antigen to be tested by 4 hemagglutination units is used as the titer of the serum antibody to determine that the titer of the serum antibody is positive for the serum to be tested. The method of the invention is simple, effective and low in cost, and can be used to analyze the positive rate of a large number of serum samples, laying a foundation for the treatment and prevention of animal diseases.

Description

A method for detecting regional distribution of swine influenza virus subtypes

technical field

The invention belongs to the technical field of animal health detection, and in particular relates to a method for detecting regional swine influenza virus subtype distribution.

Background technique

Swine Influenza Virus (SIV) belongs to Orthomyxoviridae and Influenzavirus A in classification. It is a single-stranded negative-strand RNA virus. The genome is composed of 8 independent segments of different sizes. Animal diseases, H1N1, H3N2 and H1N2 subtype swine influenza viruses are strains that are widespread in pigs and frequently cause disease in pigs. Simple SIV infection has the characteristics of high morbidity and low mortality, but swine influenza is a typical immunosuppressive disease, which will cause mixed or secondary infection of other pathogens, and cause serious economic losses.

After the Spanish flu pandemic in 1918, the influenza virus infected swine herds evolved into the classical swine influenza (H1/CS) branch. In 1976, the European H1N1 subtype avian influenza virus infected pigs and gradually replaced the CS branch to become the main branch of swine influenza virus. , that is, the Eurasian avian swine-origin influenza virus branch (H1/EA). In 2001, Hong Kong, China was the first to isolate the influenza virus of the EA branch. In addition, the H1 and H3 subtypes of seasonal influenza (HS) appeared obviously in pigs. geographical distribution. In 2009, an influenza pandemic that swept the world broke out from Mexico and spread widely among pigs in China. Through genetic evolution analysis, it was shown that the new influenza virus formed by the recombination of swine, human and avian influenza viruses was the new outbreak of pdm/ 09.

The respiratory epithelial cells of pigs have the ability to express the receptors of α-2,3 and α-2,6 influenza viruses at the same time, so pigs can be infected with human, porcine and avian influenza viruses at the same time. It is generally believed that pigs are the parent of influenza viruses. "mixer". There are multiple subtypes of influenza viruses coexisting in Chinese swine herds. Since pdm/09 infected Chinese swine herds, pdm/09 has gradually become the main epidemic branch in Chinese swine herds and began to widely exist in Chinese swine herds. Studies have shown that pdm/09 has recombined with other influenza viruses to form a new influenza virus and began to spread in pigs. Therefore, strengthening the detection of swine influenza virus is of great significance to public health. For this reason, scholars at home and abroad have done a lot of research on the diagnosis and detection of swine influenza virus, and developed detection methods such as PCR, ELISA and Real-Time PCR. These methods are time-consuming and laborious, requiring expensive experimental instruments and skilled operating skills to complete, and are not suitable for detecting the distribution of regional swine influenza viruses. The commonly used and most basic method for detecting the distribution of regional swine influenza viruses is hemagglutination and hemagglutination inhibition test, but because animal serum contains non-specific coagulation factors, it often causes false positive results and hinders the detection work. In addition, due to the heavy detection workload and many test samples, it is easy to cause disorder.

Contents of the invention

In order to solve the problem that the hemagglutination and hemagglutination inhibition tests are interfered by non-specific coagulation factors and the data analysis is difficult, the present invention intends to provide a method for detecting the distribution of regional swine influenza virus subtypes, which is simple, effective, low-cost, and suitable for The development of local testing work.

A method for detecting regional swine influenza virus subtype distribution, comprising the following steps:

Step 1. Collect serum samples, divide them into different groups according to different locations, and process each serum sample in each group. The processing steps are: add 15 μL of 8 mg/mL trypsin solution to each 30 μL serum sample, and heat at 56 °C Treat in a water bath for 30 minutes, add 60 μL of 2.3 mg/mL sodium ferrate aqueous solution and 60 μL of 3% hydrogen peroxide aqueous solution, mix well and let stand at room temperature for 15 minutes, then add 60 μL of 0.5% by mass volume percent polyethylene glycol Aqueous solution, after mixing, let stand at room temperature for 10 minutes, mix with 75 μL of PBS solution to obtain the serum to be tested; take the serum to be tested and chicken red blood cell suspension with a volume fraction of 1%, and mix the two according to the volume ratio of 1:1 , the chicken erythrocytes sink in a teardrop shape, the serum sample is successfully processed, and the serum to be tested is used in the subsequent steps;

Step 2: Add 50 μL of PBS solution to the 2-12 wells of the V-type micro-reaction plate; add 100 μL of the antigen to be tested in the 1st well, suck out 50 μL of it and place it in the 2nd well, mix well and remove from the 2nd well Aspirate 50 μL from the well and place it in the third well, serially dilute to the 11th well, and discard the final 50 μL; add 50 μL of 1% chicken red blood cell suspension to the 1st to 12th wells, and the V-type microreaction Place the plate on a micro shaker for 1-2 minutes, let it stand at 37°C for 15 minutes, observe the results, and obtain the hemagglutination unit of the antigen to be tested;

Step 3. Prepare the antigen to be tested with a concentration of 4 hemagglutination units according to the results of step 2. Add 25 μL of PBS solution to wells 2-11 of the V-type microreaction plate, and add 50 μL of PBS solution to the 12th well; Add 50 μL of the treated serum to be tested in the first well, aspirate 25 μL and place it in the second well, after mixing, aspirate 25 μL from the second well and place it in the third well, and serially dilute to the tenth well , discard the final 25 μL; add 25 μL of the antigen to be tested at a concentration of 4 hemagglutination units to wells 1-11, let stand at 37°C for 30 minutes, add 50 μL of 1% chicken red blood cells to wells 1-12 Suspension; the titer of serum antibody is taken as the titer of serum antibody at the highest dilution multiple of the tested serum that completely inhibits the antigen to be tested for 4 hemagglutination units, and it is positive when the titer of serum antibody is ≥ 40;

Step 4: Perform statistical analysis on the data obtained in Step 3, and compare whether the difference in the positive rate of the serum to be tested in different groups is significant.

As a further optimization, the preparation method of trypsin solution in step 1: add 15mL of D-Hank's solution to every 0.8g of trypsin to make a paste, then add 40mL of D-Hank's solution and stir until completely dissolved, and dilute to 100mL, adjust the pH to 7.2 with sodium bicarbonate solution, filter and sterilize, set aside.

As a further optimization, the V-type micro-reaction plate is cleaned before use. The cleaning steps are: put a cotton swab soaked in 75% ethanol in each well of the V-type micro-reaction plate and rotate it, then rinse it twice with distilled water, and then use Wash with double distilled water three times, and dry in a 37°C incubator.

As a further optimization, the 1% chicken erythrocyte suspension is the mixed blood of 3 SPF chickens, which is added to 2 times the volume of Alsever solution, mixed and shaken; 500rpm/min horizontal centrifugation for 5min, and the upper layer of Alsever is sucked out. The white blood cell layer on the surface of the liquid and the blood cell sediment was obtained to obtain the blood cell sediment; add 20 times the volume of the blood blood sediment with pH=7.2 normal saline, mix well, wash by centrifugation at 1500rpm/min for 10min, suck out the supernatant and repeat the washing 3 times , take the precipitate; mix the precipitate with normal saline at a volume ratio of 1:100 to obtain a 1% chicken erythrocyte suspension.

Beneficial effect:

The present invention uses trypsin, sodium ferrate, hydrogen peroxide and polyethylene glycol to act sequentially on serum samples, wherein trypsin degrades proteins at specific positions to degrade proteins at junctions between cells. The tension of the cytoskeleton causes the cells to separate into a spherical shape. Hydrogen peroxide interacts with the atomic oxygen released by sodium ferrate to destabilize non-specific endogenous factors, making them tend to be stable, while sodium ferrate itself is reduced, which has a certain flocculation effect, which can stabilize the remaining white blood cell fragments in serum The flocculation of non-specific endogenous factors, using the induction of polyethylene glycol, precipitates macromolecular substances, and eliminates the impact on erythrocyte agglutination. The reaction of red blood cells creates a good environment. This method is simple, effective, convenient and fast. Compared with the common removal method of non-specific coagulation factors, the effect is obvious, the agglutination rate is reduced to 0, and the interference of non-specific coagulation factors is eliminated.

detailed description

The present invention will be further explained through specific embodiments below.

A method for detecting regional swine influenza virus subtype distribution, comprising the following steps:

Step 1. Collect serum samples, divide them into different groups according to different locations, and process each serum sample in each group. The processing steps are: add 15 μL of 8 mg/mL trypsin solution to each 30 μL serum sample, and heat at 56 °C Treat in a water bath for 30 minutes, add 60 μL of 2.3 mg/mL sodium ferrate aqueous solution and 60 μL of 3% hydrogen peroxide aqueous solution, mix well and let stand at room temperature for 15 minutes, then add 60 μL of 0.5% by mass volume percent polyethylene glycol After mixing the aqueous solution, let it stand at room temperature for 10 minutes, and mix it with 75 μL of phosphate buffered saline (i.e. PBS solution) to obtain the serum to be tested; take the serum to be tested and chicken red blood cell suspension with a volume fraction of 1%, and follow the ratio of 1:1. Mix the two according to the volume ratio, the chicken red blood cells sink in a teardrop shape, the serum sample is successfully processed, and the serum to be tested is used in the subsequent steps;

Among them, the preparation method of trypsin solution: add 15mL of balanced salt solution (D-Hank's solution) to every 0.8g of trypsin to make a paste, then add 40mL of D-Hank's solution and stir until it is completely dissolved, and dilute to 100mL, adjust the pH to 7.2 with sodium bicarbonate solution, filter and sterilize, set aside.

Among them, the 1% chicken erythrocyte suspension is the mixed blood of 3 SPF chickens, added to Alsever's solution (Alsever's solution) twice the volume, mixed and shaken; 500rpm/min horizontal centrifugation for 5min, suck out the Alsever solution and the white blood cell layer on the surface of the blood cell sediment to obtain the blood cell sediment; add 20 times the volume of the blood blood sediment with pH=7.2 normal saline, mix well, wash by centrifugation at 1500rpm/min for 10min, suck out the supernatant and repeat the washing for 3 The second time, take the precipitate; mix the precipitate with normal saline at a volume ratio of 1:100 to obtain a 1% chicken erythrocyte suspension.

Step 2: Add 50 μL of PBS solution to the 2-12 wells of the V-type micro-reaction plate; add 100 μL of the antigen to be tested in the 1st well, suck out 50 μL of it and place it in the 2nd well, mix well and remove from the 2nd well Aspirate 50 μL from the well and place it in the third well, serially dilute to the 11th well, and discard the final 50 μL; add 50 μL of 1% chicken red blood cell suspension to the 1st to 12th wells, and the V-type microreaction Place the plate on a micro shaker for 1-2 minutes, let it stand at 37°C for 15 minutes, observe the results, and obtain the hemagglutination unit of the antigen to be tested;

Among them, the V-type micro-reaction plate is cleaned before use. The cleaning steps are: put a cotton swab soaked in 75% ethanol in each well of the V-type micro-reaction plate and rotate it, then rinse it twice with distilled water, and then wash it with double distilled water. Wash 3 times and dry in a 37°C incubator.

Step 3. Prepare the antigen to be tested with a concentration of 4 hemagglutination units according to the results of step 2. Add 25 μL of PBS solution to wells 2-11 of the V-type microreaction plate, and add 50 μL of PBS solution to the 12th well; Add 50 μL of the treated serum to be tested in the first well, aspirate 25 μL and place it in the second well, after mixing, aspirate 25 μL from the second well and place it in the third well, and serially dilute to the tenth well , discard the final 25 μL; add 25 μL of the antigen to be tested at a concentration of 4 hemagglutination units to wells 1-11, let stand at 37°C for 30 minutes, add 50 μL of 1% chicken red blood cells to wells 1-12 Suspension; the titer of the serum antibody is taken as the highest dilution of the tested serum that completely inhibits the antigen to be tested by 4 hemagglutination units, and it is positive when the titer of the serum antibody is ≥ 40;

Step 4: Statistically analyze the data obtained in step 3, calculate the 95% confidence interval of the positive rate of the serum to be tested at different sample sizes, and compare whether the difference in the positive rate of the serum to be tested in different groups is significant.

Determination of the effect of non-specific coagulation factor removal methods:

Use the following four methods (A, B, C, D) to treat 40 serum samples, and use 1% chicken red blood cell suspension to conduct a hemagglutination test on the above-mentioned treated serum samples to determine the removal effect of non-specific agglutination.

A: direct determination of serum samples;

B: Serum samples were treated in a water bath at 56°C for 30 minutes and then allowed to cool;

C: Determination after treatment by "trypsin-potassium periodate-glycerol" method;

D: Measured after being treated by the method of the present invention.

The measurement results are shown in Table 1 below.

Among them, "1-2 wells" means the number of fractions where agglutination occurs only in 1-2 wells; "3 wells and above" means the fractions where agglutination occurs in 3 wells and above.

The results showed that the non-specific agglutination rate of untreated serum samples was as high as 65%. After heat treatment, the non-specific agglutination rate decreased to 37.5%. The commonly used "pancreatin-potassium periodate-glycerol" The effect of the method is slightly significant, but it will still affect the hemagglutination inhibition test. After being treated with the method described in the present invention, the non-specific agglutination phenomenon disappears, and the serum sample used for the hemagglutination inhibition test will not produce false positives.

Example 1 Serological survey of influenza viruses in Jiangxi Province using a method for detecting the distribution of regional swine influenza virus subtypes

Through the serological monitoring and statistical analysis of H1 and H3 subtype swine influenza viruses in pigs in Jiangxi Province, the prevalence of influenza viruses in pigs in Jiangxi Province can be grasped.

Step 1. Collection of samples and information

From October 2012 to March 2015, a total of 2005 sera were collected covering the whole province of Jiangxi Province, including 130 samples in 2012 and 430 samples in 2013. In 2014 it was 924 and in 2015 it was 548. The serums of each city divided into districts are different, among which Fuzhou and Ji’an are the most, with 562 and 338, followed by Ganzhou, Shangrao, Nanchang, Jingdezhen, Yichun and Jiujiang, respectively 205, 172, 158, 155, 139 and 126 copies, of which Pingxiang City, Xinyu City and Yingtan City were the least, with 65, 55 and 30 copies respectively.

Step 2. Collection and processing of serum samples

Serum from pig farms in various districts and cities in Jiangxi Province was collected, and at least 15 serum samples were collected from each farm. Serum samples were processed to remove non-specific coagulation factors in the serum and stored at 4°C. The specific steps are: add 15 μL 8 mg/mL trypsin solution to each 30 μL serum sample, treat in a water bath at 56 °C for 30 minutes, add 60 μL 2.3 mg/mL sodium ferrate aqueous solution and 60 μL 3% volume percent hydrogen peroxide aqueous solution, After mixing, let it stand at room temperature for 15 minutes, add 60 μL of 0.5% polyethylene glycol aqueous solution by mass volume percentage, mix it and let it stand at room temperature for 10 minutes, and mix it with 75 μL PBS solution to obtain the serum to be tested; take the serum to be tested according to 1: The volume ratio of 1 was mixed with 1% chicken erythrocyte suspension, the chicken erythrocytes sank in a teardrop shape, the serum sample was successfully processed, and the serum to be tested was used in the subsequent steps.

Step 3. Detection and statistical analysis of serum HI antibody

First, detect the hemagglutination unit of the selected antigen, add 50 μL of PBS to the 2-12 wells of the V-type micro-reaction plate, add 100 μL of the antigen to be tested in the first well, suck out 50 μL and place it in the second well, and follow the steps of 1/ Dilute 2 times to well 11, discard the final 50 μL, add 50 μL of 1% chicken red blood cell suspension to wells 1 to 12, let stand at 37°C for 15 minutes, observe the results, and obtain the hemagglutination unit of the reference antigen .

Result judgment: the result is represented by ++++, +++, ++, +, + , -, among which:

A layer of red blood cells evenly spread on the hole wall is ++++;

A layer of red blood cells is evenly spread on the wall of the hole, but the edges are not neat, and the spread area is slightly smaller, which is +++;

Red blood cells form a ring, surrounded by small clots as ++;

Red blood cells form a small mass, but the edges are not smooth, and there are small clots around it as + ;

Red blood cells form a small circle without any small clots at the edge -.

Take ++ as the end point, that is, take the reciprocal of the highest dilution that can completely inhibit erythrocyte agglutination as the end point. Get a hemagglutination titer. Divide the hemagglutination titer by 4 to obtain the dilution factor, and dilute the standard reference antigen by this factor, which is 4 hemagglutination units.

Prepare the antigen to be tested with a concentration of 4 hemagglutination units, add 25 μL PBS to the 2-11 wells of the V-type micro-reaction plate, add 50 μL PBS to the 12th well, add 50 μL of the processed serum to be tested to the 1st well, and aspirate 25 μL was placed in the second well, and then diluted to the 10th well according to 1/2 doubling ratio, and the final 25 μL was discarded; 25 μL of the antigen to be tested at a concentration of 4 hemagglutination units was added to the 1-11 wells respectively, 37 Stand still at ℃ for 30 minutes, add 50 μL of 1% chicken red blood cell suspension to the 1-12 wells respectively; take the highest dilution factor of the serum to be tested that completely inhibits the antigen to be tested by 4 hemagglutination units as the titer of the serum antibody, and the titer of the serum antibody Antibody titer ≥ 40 is positive.

Step 4. Data statistics and analysis

The obtained data were statistically analyzed using IBM SPSS Statistics 20 and Graphpad Prism 5, and the 95% confidence intervals of the seropositive rates at different sample sizes were calculated to compare whether the differences in the seropositive rates in different groups were significant. The result is as follows:

(1) Epidemic situation of H1 and H3 subtype influenza viruses

The antibody level of influenza virus in 2005 sera collected in Jiangxi Province was detected by hemagglutination inhibition test, and the total positive rate of H1 was 34.76%, with a 95% confidence interval of 32.68%-36.89%, which was significantly higher than other subtypes of influenza virus. The seropositive rate of H3 influenza virus was 23.69%. Among H1 subtype influenza viruses, pdm/09 branch influenza virus had the highest seropositive rate of 28.73%, which was significantly higher than other branch influenza viruses, followed by H1/CS and H1/EA. The results are shown in Table 2 below.

(2) Co-infection with H1 and H3 subtype influenza viruses

If the hemagglutination inhibition test detects a positive antibody, it is considered that the pig has been infected with the influenza virus of this branch. If the antibody of two or more antigens is positive, it is considered that the pig has been infected with two or more than two branches of the influenza virus. Through statistical analysis of the experimental data, it was found that there were a large number of co-infection cases among multiple branches of the H1 subtype, the co-infection rate between pdm/09 and EA/H1N1 was 11.62%, and the co-infection rate between pdm/09 and CS The co-infection rate of EA and CS was 5.19%; the positive rate of H1 and H3 subtype influenza virus antibody was 6.78%, and the co-infection cases of H1 subtype influenza virus and H3 subtype influenza virus also appeared . The results are shown in Table 3 below.

(3) The prevalence of influenza viruses in different areas of Jiangxi Province

After statistical analysis of the serological detection results of H1 and H3 subtype influenza viruses in 11 districted cities in Jiangxi Province, it was found that the antibody positive rate of the pdm/09 branch in Shangrao City and Ji'an City was the highest, respectively 44.77% and 29.88%, and significantly higher than the antibody positive rates of other branches of influenza viruses in the region; Yingtan City, Jingdezhen City, Xinyu City and Yichun City had the highest antibody positive rates of H3/L22, respectively 50.00%, 25.16%, 43.64% and 26.62%, which were significantly higher than the antibody-positive rates of other branches of SIV in the local area; the antibody-positive rates of the pdm/09 and H1/CS branch influenza viruses in Nanchang were the highest, respectively 49.37% and 41.77%, and the two branches The difference in the positive rate of influenza virus antibody was not significant; the positive rate of influenza virus antibody in the pdm/09 and H3/L22 branches of pig populations in Jiujiang City, Ganzhou City, Pingxiang City and Fuzhou City was the highest, and was significantly higher than that of H1/EA and H1 /CS positive rate. The results are shown in Table 4 below.

(4) Epidemic situation of swine influenza virus in Jiangxi Province from 2012 to 2015

After statistical analysis of the seropositive rates of different branch influenza viruses from 2012 to 2015, it was found that the serum antibody positive rate of the pdm/09 branch influenza virus in the serum collected in Jiangxi Province in 2013 was 36.74%, which was significantly higher than that in other years, and Significantly higher than other branches and H3 subtype influenza virus antibody positive rate in the same period, it began to decline in 2014, and the positive rate decreased to 20.44% in 2015; in contrast to pdm/09, the seropositive rate of H3 influenza virus gradually increased, from 2012 to 20.44%. 4.85% in 2015 rose to 41.24% in 2015. The antibody positive rate of H1/HS was relatively stable between 2012 and 2015, while H1/CS and H1/EA reached the highest in 2014, with positive rates of 17.21% and 16.23%, respectively. The results are shown in Table 3 and Table 5 below.

Claims (5)

1. the method detecting regional swine influenza virus subtype distribution, it is characterised in that comprise the following steps：

Step one, collection blood serum sample, be divided into different group according to different location, carry out every part of blood serum sample in often group Processing, processing step is：The trypsin solution of 15 μ L 8mg/mL, 56 DEG C of water bath processing are added in every 30 μ L blood serum samples 30min, adds the Na2Fe04 aqueous solution of 60 μ L 2.3mg/mL and the aqueous hydrogen peroxide solution that 60 μ L percentage by volumes are 3%, Mix the gentle and quiet 15min that puts of rear chamber, then add the Aqueous Solutions of Polyethylene Glycol that 60 μ L mass percentage by volumes are 0.5%, mix rear chamber gentle and quiet Put 10min, mix with the PBS solution of 75 μ L, obtain serum to be checked；Take serum to be checked and the chicken red blood cell that volume fraction is 1% hangs Liquid, according to 1:Both are mixed by the volume ratio of 1, and chicken red blood cell is that teardrop shaped is sunk, and blood serum sample processes successfully, blood to be checked Clear for subsequent step；

Step 2, the PBS solution being separately added into 50 μ L in the 2-12 hole of V-type micro-reaction plate；100 μ L are added in the 1st hole Antigen to be checked, is placed in its sucking-off 50 μ L in the 2nd hole, and after mixing, from the 2nd hole, sucking-off 50 μ L is placed in the 3rd hole, carries out successively Series doubling dilution, to the 11st hole, abandons 50 final μ L；1st to 12 hole adds 50 μ L 1% chicken erythrocyte suspension, by V Type micro-reaction plate is placed on micro oscillator process 1-2min, and 37 DEG C stand 15min, observed result, obtain antigen to be checked HAU；

Step 3, according to the antigen to be checked that the result compound concentration of step 2 is 4 HAUs, at V-type micro-reaction plate Being separately added into the PBS solution of 25 μ L in 2-11 hole, the 12nd hole adds the PBS solution of 50 μ L；50 μ L are added to handle well in the 1st hole Serum to be checked, is placed in its sucking-off 25 μ L in the 2nd hole, and after mixing, from the 2nd hole, sucking-off 25 μ L is placed in the 3rd hole, carries out successively Series doubling dilution, to the 10th hole, abandons 25 final μ L；Being separately added into 25 μ L concentration in 1-11 hole is 4 HAUs Antigen to be checked, 37 DEG C stand 30 minutes, 1-12 hole is separately added into 50 μ L 1% chicken erythrocyte suspension；With complete inhibition The serum to be checked highly diluted multiple of 4 HAUs antigen to be checked is as the titer of serum antibody, serum antibody titer >=40 When for the positive；

Step 4, the data obtaining step 3 carry out statistical analysis, and relatively whether the difference of different groups of seroprevalences to be checked Significantly.

2. a kind of method detecting regional swine influenza virus subtype distribution as claimed in claim 1, it is characterised in that：Step The compound method of trypsin solution in one：Add D-Hank ' the s liquid of 15mL to be modulated into pasty state in every 0.8g pancreatin, add D-Hank ' the s liquid of 40mL stirs to being completely dissolved, and is settled to 100mL, adjusts pH to 7.2 with sodium bicarbonate solution, filtration sterilization, Standby.

3. a kind of method detecting regional swine influenza virus subtype distribution as claimed in claim 1, it is characterised in that：V-type Micro-reaction plate is carried out before use.

4. a kind of method detecting regional swine influenza virus subtype distribution as claimed in claim 1, it is characterised in that：V-type The cleaning step of micro-reaction plate is：Rotate, so in cotton swab immersed with 75% ethanol is placed in every hole of V-type micro-reaction plate Use distilled water flushing 2 times afterwards, then wash 3 times with distilled water, put in 37 DEG C of incubators and be dried.

5. a kind of method detecting regional swine influenza virus subtype distribution as claimed in claim 1, it is characterised in that：Described The chicken erythrocyte suspension of 1% is by the mixing blood of 3 SPF chickens, joins in the Alsever liquid of its 2 times of volumes, and mixing shakes up； 500rpm/min horizontal centrifugal 5min, the Alsever liquid on sucking-off upper strata and the leukocytic cream on blood cell sediment surface, obtain blood cell and sink Shallow lake thing；Adding the physiological saline of the pH=7.2 of 20 times of volumes of blood cell sediment, mixing, 1500rpm/min centrifuges 10min and enters Row washing, repeats washing 3 times, takes precipitation after sucking-off supernatant；To precipitate with physiological saline with 1:The volume ratio mixing of 100 is all Even, obtain the chicken erythrocyte suspension of 1%.