三种实时荧光RT-PCR试剂在检测甲型H1N1流感病毒中的应用

    黄宗炎 朱玉兰 刘胜牙 王佃鹏1 高朝贤 董瑞玲 黄彤文 李政良 胡孔新

    （1.深圳国际旅行卫生保健中心，深圳 518033；2.中国检验检疫科学研究院，北京100123）

    摘要 〔目的〕结合WHO公布的甲型H1N1流感病毒的检测引物和探针，探索合适的实时荧光RT-PCR试剂应用于国境口岸甲型H1N1流感病毒疑似标本的检测。〔方法〕通过对连续梯度稀释阳性对照品进行检测，从而分析AB AgPath-ID、Qiagen QuantiTect和Takara OneStep PrimeScript 3种常用实时荧光RT-PCR试剂的灵敏度、信号强度、反应效率等因素。〔结果〕3种试剂的检测下限均为1:1000倍稀释阳性对照品；AB AgPath-ID、Qiagen QuantiTect和Takara OneStep PrimeScript检测的线性斜率分别为-5.517、5.214和-4.600；信号强度上QiagenQuantiTect相对于其他2种明显偏弱。〔结论〕 为国境口岸输入性甲型H1N1流感病例的检测和监控提供了有力的技术保障。

    关键词 实时荧光RT-PCR；甲型H1N1流感病毒；灵敏度；斜率；阈值循环

    〔中图分类号〕　R511.7　〔文献标识码〕　B

    Application of  three Kinds of RT-PCRKit for Detection of Influenza A（H1N1） Viruses   HuangZongyan1, Zhu Yulan1, Liu Shengya1, Wang Dianpeng1, Gao Chaoxian1,Dong Ruiling1, Huang Tongwen1, Li Zhengliang1, Hu Kongxin2.(1.Shenzhen International Travel Health Care Center, Shenzhen518033, China; 2.Chinese Academy of Inspection and Quarantine,Beijing 100025, China)

    [Abstract]   Objective  To set up a fast and effective real time RT-PCR recipe to detectinfluenza A（H1N1）viruses emergent at frontier ports by comparingand assessing the effects of different types of real time RT-PCRkit combined with the probes and primers published by WHO.Method  Comparing and valuating the sensitivity, signalstrength and efficiency with other aspects in influenzaA（H1N1）viruses detection using three different brands of real timeRT-PCR kit named AgPath-ID, QuantiTect and One Step PrimeScriptrespectively by the method of continuous gradient dilution ofpositive influenza A（H1N1）virus RNA sample. Result The three kindsof kit had the same sensitivity at the lowest diluted positive RNAsample by 1:1000 fold; The Slope for AgPath-ID, QuantiTect and OneStep PrimeScript was -5.517，-5.214 and -4.600 respectively. As forSignal Strength, QuantiTect was much poorer than the other two.Conclusion  AgPath-ID, QuantiTect and One Step PrimeScriptcould all be used for detection of influenza A（H1N1）viruses. TheResult of numerous samples detection based on One Step PrimeScriptcombined with three panels of primers and probes WHO recommendedshowed such real time RT-PCR recipe should allow us to reactquickly and effectively during influenza A（H1N1）outbreaks atfrontier ports.

    [Key words]   Real timeRT-PCR；Influenza A（H1N1）virus；Sensitivity；Slope；Ct