CN101875979A

CN101875979A - Multi-PCR (Polymerase Chain Reaction) detection method of H5 subtype avian influenza virus variant strain

Info

Publication number: CN101875979A
Application number: CN 201010180500
Authority: CN
Inventors: 彭大新; 刘秀梵; 吴艳涛; 陈素娟; 张小荣; 徐全刚; 刘金彪
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2010-05-21
Filing date: 2010-05-21
Publication date: 2010-11-03
Anticipated expiration: 2030-05-21
Also published as: CN101875979B

Abstract

The invention relates to a method for multi-PCR (Polymerase Chain Reaction) detection of an H5 subtype avian influenza virus variant strain. In the method, RT (Reverse Transcription)-PCR amplification is carried out on corresponding target genes by using three pairs of primers, and a PCR amplified product is subjected to agarose gel electrophoresis and judged according to an electrophoresis result: if a 300bp type specific band appears, an A type influenza virus strain is judged to be; if 173bp subtype specific band appears, a H5 subtype influenza virus is judged to be; and if 559bp specific band appears, a variant strain is judged to be. The invention provides a novel method for rapid diagnosis of avian influenza and the variant strain and guidance for the selection of vaccines.

Description

The multi-PCR detection method of H5 subtype avian influenza virus variant

Technical field

Whether to bird and products thereof infect method that transmissible disease detect, be exactly a kind ofly to detect the method whether bird and products thereof infects H5 subtype avian influenza virus variant with multiplex PCR if the present invention relates to a kind of being used for.

Background technology

Bird flu is a kind of transmissible disease from respiratory system to multiple symptoms such as whole body septicemia of the bird that caused by the A of orthomyxoviridae family type influenza virus.High pathogenic avian influenza is to be caused by the H5N1 subtype avian influenza virus, and its M ﹠ M is high, is one of main deadly infectious disease of harm bird, is decided to be legal circular transmissible disease by International Office of Epizootics.China mainly takes compulsory immunization to high pathogenic avian influenza and slaughters the prevention and control strategy that combines at present, and the existence of immune pressure can cause virus to morph, and causes the generation of variant, causes clinical immuning failure, is necessary to carry out epidemiology survey work.In the end of the year 2008, H5N1 subtype highly pathogenic avian influenza virus variant appears in the East China, has caused huge strike for local aviculture.Therefore, set up a kind of technology that detects the H5 subtype avian influenza virus fast and accurately, can in time determine pathogenic agent, help people and in time take effective prevention and control measure, plant's financial loss is significant for reducing.

In recent years, the RT-PCR method is used in the detection of bird flu widely, and this method can remedy the deficiency of the classical detection method of bird flu, and has good specificity and susceptibility.Report such as nineteen ninety-five Wright is used the RT-PCR method and is distinguished A, B, the bird flu of C type, China Liu Yan etc. has set up the multiplex RT-PCR method of differentiating H5 and H9 hypotype AIV, foundation such as Xie Zhixun etc. have set up the multiplex RT-PCR method of differentiating H5 and H7 hypotype AIV, Xie Z have also been optimized the multiple RT-PCR that detects A type AIV and can distinguish H5, H7 and H9 hypotype AIV simultaneously.

Summary of the invention

The present invention further explores the RT-PCR method that detects AIV, has designed the special primer of the variant of type (A type), hypotype (H5) and clade7 at avian influenza virus, has set up multiple RT-PCR detection method.This method not only can Rapid identification A type influenza virus in same RT-PCR reaction system, and can determine whether it is the avian influenza virus of H5 hypotype and variant.

The present invention chooses the conservative relatively M gene design type specificity primer of avian influenza virus; Choose the conservative a pair of primer of zone design of HA genetic comparison, in order to determine whether the being H5 subtype avian influenza virus; Whether it mainly makes a variation variant and mainly concentrates on the HA gene, so just choose and the bigger primer of zone design of strain variation in the past, be the variant of clade7 in order to detect.

The technical solution adopted in the present invention is: try to extract son or blood or organ-tissue or the allantoic fluid full geneome RNA from tracheae or the cloaca of fowl to be checked, be template again with RNA, with AIV stromatin (M), hemagglutinin (H5) gene is that target gene design mode Auele Specific Primer, hypospecificity primer and the primer of identifying variant carry out the RT-PCR amplification, pcr amplification product is judged according to electrophoresis result then through agarose gel electrophoresis;

M1 5′-ATTGGGACTCATCCTAACTCTA-3′

M2 5′-CGTCAACATCCACAGCACTC-3′

HA1 5′-ATRCCATTYCACAACATACACC-3′

HA2 5′-TCCYTGCCATCCTCCCTC-3′

HA3 5′-TAATACATACCCACCAATAAAGGTGA-3′

HA2 5′-TCCYTGCCATCCTCCCTC-3′

Wherein, R is A or G; Y is C or T.

Occur in the electrophoretogram 300bp the type specific band be A type influenza strain; Occur 173bp the subtype sepcific band be H5 subtype influenza virus, what the 559bp specific band occurs is variant.

Utilize 3 pairs of primers of institute's synthetic that corresponding target genes is carried out the RT-PCR amplification, set up multiple RT-PCR detection method, this method not only can be identified A type influenza virus and H5 subtype avian influenza in 1 reaction system, and can determine whether H5 subtype avian influenza virus simultaneously into clade 7 variants, and the method for classical chicken embryo propagation cannot determine whether isolating bird flu strain is variant, so this method has important use to be worth at aspects such as quick screening of clinical sample and the evaluations of AIV hypotype.For the quick diagnosis of bird flu and variant provides new method, for the selection of vaccine provides directive function.

Description of drawings

Fig. 1 is a multiple RT-PCR specificity test-results

M:100bp Marker wherein; 1:H1AIV; 2:H3AIV; 3:H4AIV; 4:H5clade 2.3.4AIV; 5:H5clade 7AIV; 6:H6AIV; 7:H8AIV; 8:H9AIV; 9:H10AIV; 10:H11AIV; 11:NDV; 12:IBV.

Fig. 2 is multiple RT-PCR sensitivity test result

M:100bp Marker wherein; 1:460ng/ μ l AIV RNA; 2:46ng/ μ l AIV RNA; 3:4.6ng/ μ l AIVRNA4:0.46ng/ μ l AIV RNA.

Fig. 3 is a multiple RT-PCR broad spectrum test-results

M:100bp Marker wherein;

1：A/chicken/Huadong/4/2008；2：A/chicken/Huadong/5/2008

3：A/chicken/Huadong/2/2009；4：A/chicken/Huadong/1/2009；

5：A/chicken/Huadong/5/2009；6：A/chicken/Huadong/3/2008；

7：A/chicken/Huadong/2/2008；8：A/goose/Huadong/3/2009.

Embodiment

Concrete technical scheme of the present invention is as follows:

(1) primer design

According to experiment purpose, through homology relatively, select the appropriate area of AIV stromatin (M), hemagglutinin (H5) gene, respectively the primer of design mode Auele Specific Primer, hypospecificity primer and evaluation variant.Primer sequence is as follows:

Annotate: annex base code R=A/G Y=C/T

(2) extraction of viral RNA

Get the centrifuge tube that 1.5mL DEPC handled, add H1 hypotype AIV, H3 hypotype AIV, H4 hypotype AIV, H5 hypotype clade2.3.4 AIV, H6 hypotype AIV, H8 hypotype AIV, H9 hypotype AIV, H10 hypotype AIV, H11 hypotype AIV, NDV, IBV allantoic fluid 100 μ l and with the PBS of sterilization the organ-tissue samples such as tracheae of doubtful AIV are ground to form the suspension 100 μ l that dilute at 1: 5, add 300 μ l sex change liquid again, continue to add 30 μ l 2M sodium-acetates.Put upside down centrifuge tube 4-5 time repeatedly, to mix.Add phenol/chloroform/primary isoamyl alcohol mixed solution 300 μ l, carefully mix centrifuge tube 3-5 time, acutely rocked again 10 seconds, place static 15min on ice.12000 rev/mins of 4 ℃ of centrifugal 20min carefully shift the centrifuge tube of supernatant in an other clean 1.5mL.Add isopyknic Virahol, leave standstill 5min at least, precipitated rna in-20 ℃.12000 rev/mins of 4 ℃ of centrifugal 10min.Abandon supernatant, add 75% ice ethanol, gentle puts upside down centrifuge tube 3-5 time, 12000 rev/mins of 4 ℃ of centrifugal 5min repeatedly.Abandon supernatant, dry back adds 10 μ lDEPC treating water, dissolving RNA ,-70 ℃ of preservations.

(3) RT-PCR amplicon virus gene fragment

Get 1 μ L cDNA template and carry out pcr amplification, system is as follows: 2.5 μ L10 * buffer, 1.5 μ L Mg ²⁺, 0.5 μ L dNTP, each 1 μ L of primer M1, M2, HA11 μ L, each 0.5 μ L (concentration is 50pmol/ μ L) of HA2 and HA3,14 μ L ultrapure waters, 0.5 μ L TaqDNA polysaccharase.Response procedures: pre-denaturation temperature be 94 ℃ 5 minutes, then by 94 ℃ of 45s, 58 ℃ of 30s, 72 ℃ of 30s carry out 35 circulations, last 72 ℃ are extended 10min.

(4) evaluation of pcr amplification product

Get pcr amplification product 10 μ l, point sample is in 1.5% sepharose (containing 0.5 μ g/mL ethidium bromide), with 100bp Laddermarker as standard reference, visible multiple RT-PCR detects the type specific band that 300bp all appears in all A type influenza strains behind the electrophoresis, the subtype sepcific band of 173bp all appears in H5 subtype influenza virus, the specific band of 559bp appears in variant (swimming lane 5), and this method detects NDV simultaneously, IBV result is all negative.These results show that this method has good specificity (Fig. 1).

(5) multiple RT-PCR sensitivity test result

With ultraviolet spectrophotometer AIV RNA is carried out quantitatively, and do limiting dilution, detect, determine that it is limited to 4.6ng/ μ l viral RNA (Fig. 2) under detecting with multiplex RT-PCR method.

(6) multiple RT-PCR broad spectrum test-results

Select the H5N1 subtype avian influenza virus of 6

strain Clade

7 and 2 strain Clade2.3.4, through RT-PCR, three specific bands all appear in 6 variants, and type specific band and hypospecificity band (Fig. 3) only appear in 2 strain Clade2.3.4H5N1 subtype avian influenza virus.

Claims

1. the multi-PCR detection method of a H5 subtype avian influenza virus variant, it is characterized in that trying to extract son or blood or organ-tissue or the allantoic fluid full geneome RNA from tracheae or the cloaca of fowl to be checked, be template again with RNA, with AIV stromatin (M), hemagglutinin (H5) gene is that target gene design mode Auele Specific Primer, hypospecificity primer and the primer of identifying variant carry out the RT-PCR amplification, pcr amplification product is judged according to electrophoresis result then through agarose gel electrophoresis;

M1 5′-ATTGGGACTCATCCTAACTCTA-3′

M2 5′-CGTCAACATCCACAGCACTC-3′

HA1 5′-ATRCCATTYCACAACATACACC-3′

HA2 5′-TCCYTGCCATCCTCCCTC-3′

HA3 5′-TAATACATACCCACCAATAAAGGTGA-3′

HA2 5′-TCCYTGCCATCCTCCCTC-3′

Wherein, R is A or G; Y is C or T.

2. the multi-PCR detection method of H5 subtype avian influenza virus variant according to claim 1 is characterized in that: be with the PBS that sterilizes the sample that is extracted to be ground to form the suspension that dilutes at 1: 5, extract full geneome RNA with the Trizol method again.

3. the multi-PCR detection method of H5 subtype avian influenza virus variant according to claim 1 is characterized in that: electrophoresis occur 300bp the type specific band be A type influenza strain; Occur 173bp the subtype sepcific band be H5 subtype influenza virus, what the 559bp specific band occurs is variant.