应用PCR-RFLP技术进行乙型流感病毒鉴别的实验研究  

Differentiation of influenza B viruses by PCR-RFLP assay

作　　者：姚栩[1] 张彩云[1] 陈智伟[1] 杨建娜[1] 郑能雄[1] 

机构地区：[1]福州市疾病预防控制中心,福州350004

出　　处：《中国卫生检验杂志》2007年第11期1948-1950,共3页Chinese Journal of Health Laboratory Technology

基　　金：福建省卫生系统学术技术带头人项目资助(2005)

摘　　要：目的:应用聚合酶链反应(PCR)连接的限制性片段长度多态性(RELP)技术进行乙型流感病毒及其巴拿马株与维多利亚株两大谱系的鉴别。方法:根据GenBank中登录乙型流感病毒血凝素(HA)区域的核苷酸序列,应用软件进行序列比对,根据HA基因的保守区设计、筛选引物,采用逆转录-聚合酶链反应(RT-PCR)扩增流感病毒HA基因,用限制性内切酶H indⅢ酶切扩增产物并进行琼脂糖凝胶电泳,同时对检测方法进行特异性和灵敏性实验。结果:该方法对乙型流感病毒有特异性,与甲1和甲3型流感病毒无交叉反应。维多利亚株和巴拿马株HA基因均能被特异性引物所扩增,扩增产物经H indⅢ酶切后,乙型流感病毒巴拿马株在电泳谱上出现603 bp与242 bp的两个可见片段,而维多利亚株因无H indⅢ酶切位点,仍呈872 bp的一个片段。该方法检测敏感度可达0.1 TC ID50。结论:该方法是一种灵敏度高、特异性强的乙型流感病毒维多利亚株与巴拿马株鉴别的方法。Objective： To differentiate the Yamagata strain and Victoria strain of influenza B. Methods：The hemagglutimin （HA） genes of influenza B viruses down - loaded from Genbank were aligned by using the biologic software and the specific primers were designed and selected in the consered regions of HA. HA genes of Yamagata strain and Victoria strain of influenza B were amplified by RT - PCR. The speciflc amplified products were analyzed by restriction endonuclease Hind Ⅲ digestion and agarroso gel electrophoresis. The sensitivity and specificity of the assay were evaluated. Results：The specificity of this assay was high with influenza B and had no cross reactions with influenza A1 and A3. The primer pair used in RT - PCR was specific for both Yamagata strain and Victoria strain. The amplified product of Yamagata strain could be cut into two fragments by Hind Ⅲ ,which were 603 bp and 242 bp,respectively. The amplified fragment was still 872 bp in Victoria strain because of no cleavage site for Hind Ⅲ. The sensitivity of this assay was 0. 1 TCID50. Conclusion：PCR - RFLP is a sensitive and specific method for differentiation of Yamagata strain and Victoria strain of. influenza B viruses.

关 键 词：乙型流感病毒 限制性片段长度多态性 聚合酶链反应 鉴别 

分 类 号：R373.13[医药卫生—病原生物学]