甲型H1N1流感病毒RNA检测方法的建立及临床应用

Establishment of influenza A H1N1 virus RNA determination method and its clinical application

目的建立检测甲型H1N1流感病毒RNA的实时荧光定量逆转录聚合酶链反应(RT-PCR)方法,并探讨其临床应用价值。方法根据甲型H1N1流感病毒HA基因和NA基因的核苷酸序列和GenBank数据库中的核苷酸序列设计检测特异性引物和TaqMan探针,优化反应体系和反应条件,建立检测甲型H1N1流感病毒RNA的实时荧光定量RT-PCR方法,并对方法的敏感性、特异性和重复性进行评价。采用该方法检测21份鼻咽拭子样本的甲型H1N1流感病毒RNA,并与鸡胚培养法进行比较。结果采用实时荧光定量RT-PCR检测甲型H1N1流感病毒RNA结果为阳性,H1~H16流感病毒、季节性流感病毒、猪流感病毒RNA检测结果均为阴性。实时荧光定量RT-PCR的检测敏感性为10^(3)拷贝/μL RNA分子,标准曲线r2=0.998。阳性RNA标准品重复检测的循环阈值(Ct)批内、批间变异系数(CV)均<10%,阴性标准品[焦碳酸二乙酯(DEPC)水]重复检测的Ct值均<0,重复性良好。21份鼻咽拭子样本中有2份样本甲型H1N1流感病毒检测结果为阳性,其他样本均为阴性,与鸡胚培养法的符合率为100%。结论建立的检测甲型H1N1流感病毒RNA的实时荧光定量RT-PCR方法操作简单、耗时短、特异性较强、敏感性较高,可在临床广泛使用。

Objective To establish a real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-PCR)for influenza A H1N1 virus RNA,and to investigate its clinical application value.Methods Specific primers and TaqMan probes were designed according to nucleotide sequences of HA and NA genes of influenza A H1N1 virus and nucleotide sequences in GenBank database.The system and conditions in the experimental reaction were improved,and the RT-PCR was established for influenza A H1N1 virus RNA.Its sensitivity,specificity and repeatability were evaluated.Totally,21 samples were collected,all of which were nasopharyngeal swab samples.A comparative study was conducted with chicken embryo culture method.Results The results of RT-PCR were different among different viruses.The RNA of influenza A H1N1 virus was positive,and the RNA of H1-H16 influenza viruses,seasonal influenza virus and swine influenza virus was negative.The sensitivity was 10^(3) copies/μL RNA molecule,and the standard curve r2 was 0.998.The cycle threshold(Ct)value within-run and between-run coefficients of variation(CV)of the positive RNA standard were<10%,and the Ct value of the negative RNA standard[diethyl pyrocarbonate(DEPC)]was<0,and the repeatability was good.In the 21 samples,only 2 samples were positive,and the remaining samples were all negative.Compared with chicken embryo culture method,the consistency was 100%.Conclusions The established RT-PCR for influenza A H1N1 virus RNA is simple,time consuming,specific and sensitive,which can be widely used in clinical practice.