甲型H1N1流感病毒RNA检测方法的建立及临床应用

doi:10.3969/j.issn.1673-8640.2021.07.013

摘要/Abstract

摘要：

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10³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。

关键词: 甲型H1N1流感病毒, 实时荧光逆转录聚合酶链反应, 检测方法, 临床应用

Abstract:

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 103 copies/μL RNA molecule,and the standard curver ² 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.

Key words: Influenza A H1N1 virus, Real-time fluorescence quantitative reverse transcription-polymerase chain reaction, Determination method, Clinical application

中图分类号:

R446.1

唐钧, 曹红梅. 甲型H1N1流感病毒RNA检测方法的建立及临床应用[J]. 检验医学, 2021, 36(7): 738-742.

TANG Jun, CAO Hongmei. Establishment of influenza A H1N1 virus RNA determination method and its clinical application[J]. Laboratory Medicine, 2021, 36(7): 738-742.

图/表 3

图1 基于HA和NA探针及引物的扩增动力学曲线注：（a）HA基因;（b）NA基因。

图2 实时荧光定量RT-PCR检测RNA标准品的敏感性试验结果及标准曲线注：（a）敏感性试验中RNA标准品的扩增曲线;（b）标准曲线（r2=0.998）。

图3 RNA阳性标准品及阴性标准品批内重复检测的扩增动力学曲线

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