流感病毒型别及亚型的多重RT-PCR方法快速检测  

Multiplex RT-PCR for the rapid detection of influenza virus types and subtypes

作　　者：卢亦愚[1] 严菊英[1] 茅海燕[1] 孙琰[1] 周敏[1] 史雯[1] 

机构地区：[1]浙江省疾病预防控制中心病毒研究所,310009

出　　处：《中华实验和临床病毒学杂志》2005年第3期252-255,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：浙江省自然科学基金资助项目(398498)

摘　　要：目的为适应流感疫情监测中快速诊断的需要,建立敏感特异的流感病毒多重逆转录PCR(MRTPCR)检测方法。方法对甲1型(H1N1)、甲3型(H3N2)、乙型流感病毒的血凝素(HA)基因保守区域分别设计引物进行MRTPCR。另设计了两对引物对H1N1和H3N2亚型流感病毒的神经氨酸酶(NA)N1、N2作亚型判断。结果MRTPCR可特异性检测出各型流感病毒的目的片段,相互间无交叉反应。二次PCR反应后对H1N1、H3N2流感病毒的检测灵敏度可达0.10TCID50/50μl以下,对乙型流感病毒的检测灵敏度可达0.01TCID50/50μl以下。应用此方法也可特异性地检测出H1N1和H3N2流感病毒的NA基因。结论用MRTPCR从临床患者含漱液标本中检出相关流感病毒的灵敏度要高于用狗肾传代细胞(MDCK)或鸡胚分离的灵敏度,达到了快速、敏感、正确检测流感病毒及其亚型的目的。Objective To establish a sensitive and specific multiplex RT-PCR （MRT-PCR） for the simultaneous detection of influenza virus types and subtypes. Methods Primers were designed from highly conserved region of the hemagglutinin （HA） gene of influenza H1N1 ,H3N2 and B virus and MRT-PCR was performed, Additional two pairs of primers were designed to determine the N1 and N2 subtypes of neuramidinase （NA） of influenza H1N1 and H3N2 virus. Results The fragments of HA gene of all types of influenza virus were amplified and there was no cross reaction. The sensitivity of detection of influenza H1N1 and H3N2 virus was 0. 10 TCID50/50 μl by the second PCR and that was 0. 01 TCID50/50 μl for influenza B virus. The NA gene of influenza H1N1 and H3N2 virus was also amplified by this method. Conclusion The sensitivity of detection of influenza virus from clinical patients＇ throat washing specimens by MRT-PCR was higher than that by MDCK cell culture or egg embryo isolation. This method was highly sensitive and timelv for deteetion of influenza virus tvpes and subtypes.

关 键 词：正粘病毒科 逆转录聚合酶链反应 基因 乙型流感病毒 H3N2亚型 PCR方法 快速检测 MRT 多重 检测灵敏度 

分 类 号：R446.5[医药卫生—诊断学]