人感染高致病性禽流感病毒H5N1核酸检测方法的建立及应用

目的 建立人感染高致病性禽流感病毒H5N1的核酸检测方法,用于人感染高致病性禽流感病毒疑似病例临床标本的检测.方法 针对甲型流感病毒保守基因M设计RT-PCR和real-time PCR引物检测是否为甲型流感病毒,同时针对H5N1禽流感病毒设计针对H5和N1基因的特异性RT-PCR和real-time PCR引物作亚型检测,建立禽流感H5N1病毒RT-PCR和real-time PCR检测方法.结果 本研究建立的RT-PCR和real-time PCR方法可以特异性地检测H5N1病毒,并且与人流感病毒H1、H3没有交叉反应.RT-PCR检测方法灵敏度可到1TCID50,real-time PCR灵敏度可达0.01TCID50.利用上述方法检测人感染高致病性禽流感病毒H5N1疑似病例临床标本,从42例不明原因肺炎病例中检测出阳性标本13例.结论 本研究建立的RT-PCR和real-time PCR方法可以用于人感染高致病性禽流感病毒H5N1临床标本的实验室检测.

Development of methods for detection of H5N1 RNA from human clinical specimens

Objective To establish a method for H5N1 RNA detection and laboratory diagnosis of suspected human avian influenza (H5N1) virus infected cases. Methods The typing and sub-typing primers were designed according to M and H5 and N1 gene respectively, and the RT-PCR and real-time PCR were developed by using these primers. Results The RT-PCR and real-time PCR could be used for H5N1 detection specifically, and there was no cross reaction with other influenza subtypes such as H1 and H3.The sensitivity for RT-PCR and real-time PCR was 1 TCID_ 50 and 0.01 TCID_ 50 respectively. Thirteen laboratory confirmed human H5N1 cases were detected from 42 suspected cases by using these methods. Conclusion The established RT-PCR and real-time PCR methods can be used for laboratory detection of suspected human H5N1 cases.