焦测序法检测禽流感病毒

以焦测序技术为检测平台,在研究禽流感病毒基因特性的基础上,建立一种检测禽流感病毒及确定其是否为高致病性禽流感病毒的序列测定法。首先,选择一段保守的M基因序列及一段包含裂解位点的HA基因序列为研究对象,采用聚合酶链反应(polymerase chain reaction,PCR)扩增技术初步判断其是否为禽流感病毒及病毒亚型;然后采用焦测序法检测目的片段序列;最后,对焦测序法检测序列进行分析,从基因序列上判断其是否为禽流感病毒,并进一步判断病毒的亚型以及是否为高致病性禽流感病毒。研究结果表明,当焦测序反应中三磷酸酰苷双磷酸酶(Apyrase)的浓度为1.6U/mL时,能有效抑制错误信号的产生;当Klenow的浓度为90U/mL时,可读序列长度为33个碱基。采用优化的焦测序反应体系测定了4个样本,其中1个样本被判断为H5N1亚型禽流感病毒,具有潜在的高致病性;另外3个样本为H9N2型禽流感病毒,具有低致病性。本方法具有准确、快速和实时检测等优点。

Detection of Avian Influenza A Virus Using Pyrosequencing

Pyrosequencing was developed for rapidly detecting avian influenza A virus and predicting the virulence.The avian influenza A virus and its subtype were determined by PCR on a species-specific sequence of the M gene and a subtype-specific sequence of the HA gene containing cleavage site,respectively.The amplicons were then analyzed by the pyrosequencing to further validate the results obtained by PCR.By optimizing the concentrations of Apyrase and Klenow,the results indicated that the false signals were effectively suppressed using 1.6 U/mL Apyrase,and the number of accurately readout bases was 33 for 90 U/mL Klenow.Based on the optimized pyrosequencing system,four specimens including an H5N1 subtype with high pathogenicity and three specimens of avian influenza A H9N2 subtype with low pathogenicity were confirmed.This method is accurate,fast,and can be used for identifying the pathogenicity of avian influenza A virus efficiently.