The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR
The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR
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DOI：
10.1016/j.jcv.2010.03.012
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发表时间：
2010-06-01
影响因子：
8.8
通讯作者：
Jarman, Richard G.
中科院分区：
文献类型：
--
作者：
Klungthong, Chonticha;Chinnawirotpisan, Piyawan;Jarman, Richard G.
背景：为应对2009年H1N1大流行，美国疾病预防控制中心和世卫组织迅速开发并分发了一种实时RT-PCR试剂盒，用于在临床样本中检测该菌株。世卫组织swH1引物和探针对东南亚2009年甲型H1N1流感毒株的敏感性存在差异。目的：探讨SEA中2009年甲型H1N1流感病毒引物与探针模板不匹配导致实时RT-PCR敏感性降低的原因。研究设计：选取37例swH1阳性样本，根据实时RT-PCR结果分为敏感组和不敏感组，进行血凝素（HA）基因测序。检测病毒swH1引物与探针结合区的序列是否不匹配。系统发育分析研究了这些病毒的多样性。重新设计引物和探针以匹配我们的每个序列，并测试以确定对灵敏度的影响。结果：从高和低敏感性患者分离的病毒HA测序显示，探针第3碱基的单个不匹配降低了23/37病毒的敏感性。同源引物和探针提高了灵敏度（平均差异为4.66,P < 0.0001）。系统进化树显示，本研究中的病毒聚为两组，与RT-PCR敏感性一致。结论：研究结果表明，在东南亚地区传播的新型H1N1病毒至少有两种变异和HA基因突变对WHO swH1引物和探针的检测有直接影响。Elsevier B.V.出版
Background: In response to the 2009 H1N1 pandemic the US CDC and WHO rapidly developed and distributed a real-time RT-PCR kit to detect this strain in clinical samples. The results from the WHO swH1 primer and probe set exhibited diverse sensitivities for the 2009 influenza A/H1N1 strains in Southeast Asia (SEA).Objective: Investigate the primer and probe-template mismatches among the 2009 influenza A/H1N1 strains in SEA that reduced the real-time RT-PCR sensitivity.Study design: Thirty-seven swH1 positive samples categorized into sensitive and insensitive groups based on real-time RT-PCR results were selected for hemagglutinin (HA) gene sequencing. The sequence in swH1 primer and probe binding regions of the viruses was examined for mismatches. Phylogenetic analysis was performed to investigate the diversity among these viruses. Primers and probe were redesigned to match each of our sequences and tested to determine the impact on sensitivity.Results: HA sequencing of the viruses isolated from patients with high and low sensitivities revealed that a single mismatch at the 3rd base of the probe reduced sensitivity in 23/37 viruses. Homologous primers and probes increased the sensitivity (mean difference 4.66 Ct P < 0.0001). Phylogenetic tree revealed that the viruses in this study clustered into two groups, coinciding with RT-PCR sensitivity.Conclusion: Results obtained indicate that at least two variants of the novel H1N1 transmitting in SEA and the mutations in HA gene have a direct effect on the detection by using WHO swH1 primer and probe set. Published by Elsevier B.V.