Design and Validation of Real-Time Reverse Transcription-PCR Assays for Detection of Pandemic (H1N1) 2009 Virus
Design and Validation of Real-Time Reverse Transcription-PCR Assays for Detection of Pandemic (H1N1) 2009 Virus
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DOI：
10.1128/jcm.01103-09
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发表时间：
2009-11-01
影响因子：
9.4
通讯作者：
Preiksaitis, Jutta K.
中科院分区：
文献类型：
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作者：
Pabbaraju, Kanti;Wong, Sallene;Preiksaitis, Jutta K.
追踪有可能引起大流行的新型流感病毒，如2009年H1N1大流行病毒，是一项公共卫生重点。2009年H1N1流感大流行病毒于2009年4月在墨西哥首次发现，并在短时间内传播到世界各地。需要经过充分验证的诊断工具，以便快速、敏感和特异性地检测和跟踪这种病毒。建立了3种实时反转录PCR （RT-PCR）扩增和检测2009年H1N1大流行病毒的检测方法，并将其性能特征与其他已发表的诊断检测方法进行了比较。来自加拿大艾伯塔省的39个样本被确认为2009年H1N1大流行病毒阳性，另外6个样本被证实为甲型流感病毒阳性，但无法通过已公布的季节性H1/H3流感病毒测定方法进行分型。产品的扩增和直接测序被认为是病例鉴定的“金标准”。当5 μ l作为模板时，新的检测方法是敏感的，并且能够在10（-6）稀释度为4 × 10(6) 50%的组织培养感染剂量/ml时重复检测病毒。它们显示出100%的特异性，并且与其他呼吸道病毒或季节性甲型流感病毒亚型没有交叉反应。2009年H1N1流感大流行病毒不同模板浓度交叉周期阈值的变异系数为
Tracking novel influenza viruses which have the potential to cause pandemics, such as the pandemic (H1N1) 2009 virus, is a public health priority. Pandemic (H1N1) 2009 virus was first identified in Mexico in April 2009 and spread worldwide over a short period of time. Well-validated diagnostic tools that are rapid, sensitive, and specific for the detection and tracking of this virus are needed. Three real-time reverse transcription PCR (RT-PCR) assays for the amplification and detection of pandemic (H1N1) 2009 virus were developed, and their performance characteristics were compared with those of other published diagnostic assays. Thirty-nine samples confirmed to be positive for pandemic (H1N1) 2009 virus from Alberta, Canada, and six additional samples that were positive for influenza A virus but that were not typeable by using published seasonal influenza H1/H3 virus assays were available for this validation. Amplification and direct sequencing of the products was considered the "gold standard" for case identification. The new assays were sensitive and able to reproducibly detect virus in a 10(-6) dilution of 4 x 10(6) 50% tissue culture infective doses/ml when 5 mu l was used as the template. They showed 100% specificity and did not cross-react with other respiratory viruses or seasonal influenza A virus subtypes. The coefficient of variation in crossing cycle threshold values for the detection of different template concentrations of pandemic (H1N1) 2009 virus was