焦磷酸测序技术在确证猪甲型H1N1流感病毒中的应用

目的本研究旨在通过对猪甲型H1N1流感病毒进行序列信息分析的基础上,利用焦磷酸测序技术建立一种快速、简单地确证猪甲型H1N1流感病毒的方法。方法通过序列信息比对,设计H1HA和N1NA基因保守区段的扩增引物及测序引物。从感染猪甲型H1N1病毒的鸡胚尿囊液中提取病毒RNA,RT-PCR扩增目的基因片段,采用焦磷酸测序技术(PSQ)针对HA基因和NA基因进行保守核苷酸区段的测序分析。利用扩增引物与其他猪源病毒进行特异性试验,利用测序引物进行重复性试验。将该方法与病毒分离和荧光定量RT-PCR方法做临床样品的平行检测,并比较结果。结果通过序列信息比对寻找到表征H1N1亚型的核苷酸保守区段,经焦磷酸测序后能进一步确证毒株的序列信息为猪甲型H1N1流感病毒。特异性试验表明,不与其他猪源病毒发生交叉反应;重复性试验表明,重现性为100%。对221份临床样品检测表明,病毒分离鉴定与焦磷酸测序方法结果符合率为96.8%,与TaqMan荧光定量方法检测结果符合率90.3%。经统计学分析,焦磷酸测序确证与病毒分离鉴定在检测临床样品上,两者差异不显著。结论基于序列分析的焦磷酸测序技术可以作为进一步确证方法使用。

Application of Pyrosequencing Technology in Confirmation of Swine Influenza A H1N1 Virus

Objective To establish a new molecular method for simultaneoulsly rapid detection and confirmation of subtype H1N1 swine influence virus by using sequence analysis combined with pyrosequencing technology.Methods According to the published sequences in GenBank,two amplification primers and one sequencing primer were designed from conserved regions of the H1HA and N1NA genes.Total SIV RNA was extracted from chorioallantois fluid infected with H1N1 swine influence virus,and RT-PCR were developed for conserved regions of HA and NA gene respectively.The RT-PCR products were pysrosequenced by pyrosequencing.The specificity and the reproductivity were tested by using the amplification primer andthe sequencing primer.The method was used to detect the 221 clinical samples and compared to the viruse isolation and real-time PCR.Results Two nucleotide segments which characterized by H1N1 were abtained by sequence analysis.And then the two segments were confirmed as H1N1 swine influence virus by pyrosequeing.The reproducibility result showed 100% in agreement and no cross-reaction was discovered with other swine disease vaccines.The method was used to detect the 221 clinical samples and the results were 96.8% in agreement with those of virus isolation in embryonated eggs.Compared to the Taqman fluorescent quantitation method,they showed 90.3% agreement.Conclusion Pyrosequencing technology based on sequences analysis can be used to confirm the subtype H1N1 swine influenza A virus.