用逆转录-聚合酶链反应鉴别甲型和乙型流感病毒感染

Identification of influenza viruses types A and B by reverse transcription polymerase chain reaction

目的 建立快速、特异、有效的鉴别甲、乙型流感病毒的方法。方法 根据编码甲、乙型流感病毒膜蛋白M基因的核苷酸序列设计两对引物 ,将这两对引物用于同一逆转录 聚合酶链反应(RT PCR) ,甲、乙型毒株的扩增产物分别为 5 0 6bp和 2 40bp ,根据这些产物在 1.2 %的琼脂糖凝胶电泳上的大小 ,即可鉴别所测毒株的型别。结果 用这一方法对我国 2 4株甲型和 5株乙型流感病毒分离株进行型别鉴定 ,分型结果与血凝抑制试验和核苷酸测定结果完全相符。结论 用上述根据M基因序列设计的RT PCR方法鉴别甲、乙型流感病毒具有快速、简便、特异等特点 ,适用于流感病毒型别鉴定 。

Objective To establish a rapid, specific and effective technique for differentiating types A and B of influenza viruses in clinical isolates. Methods Two primer sets were designed according to the nucleotide sequences of Matrix genes of influenza A and B. First strand of viral cDNA was synthesized from viral RNA by using Oliogo d (T) 18 . PCR was performed with a mixture of the two primer sets specific for influenza types A and B, respectively. Amplified products were visualized in 1.2 % agarose gel containing ethidium bromide. Types A and B were differentiated by the sizes of the products (506bp for type A and 240bp for type B). Results Twenty nine isolates from clinical specimens which have been typed by hemagglutination inhibition (HAI) were identified by the method described above and the results of identified types of influenza viruses were consistent with those obtained with the HAI test and sequencing of the HA genes. Conclusion The RT PCR technique described here is simple, convenient, specific and therefore can be used for detection and identification of influenza viruses A and B in clinical isolates. It can provide a useful alternative to existing methods of influenza identification and offer the basis for clinical diagnosis, prevention and timely specific treatment.