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首页> 外文期刊>Journal of Clinical Microbiology >Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses
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Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

机译：实时逆转录-PCR检测人类鼻病毒的全面检测。

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Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5′ noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 × 105 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRV-culture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEV-positive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.
机译：人类鼻病毒（HRV）是呼吸系统疾病的重要贡献者，但其医疗保健负担仍不清楚，这主要是因为缺乏确定其因果关系的灵敏，准确和方便的方法。为了解决这个问题，我们开发并临床验证了实时逆转录PCR（RT-PCR）测定法的敏感性和特异性，该测定法针对从所有100种目前公认的HRV原型菌株和最近的85种菌株获得的序列定义的病毒5'非编码区循环场分离物。该测定法成功扩增了所有测试的HRV，并且可重复检测50个HRV RNA转录本拷贝，动态范围超过7个对数。相反，在低于每个反应5×10 5 的浓度下，未检测到与HRV引物和探针组具有最大序列同源性的人类肠道病毒68（HEV68）定量RNA转录本。培养的HRV或HEV呈阳性的111个编码呼吸道样本的核酸提取物分别通过HRV实时RT-PCR分析和两个使用不同室内HRV / HEV RT-PCR分析的独立实验室进行了测试。通过实时RT-PCR测定正确鉴定了87个HRV培养阳性标本，在24个HEV阳性样品中有4个为HRV阳性。随后在这四个样本中鉴定出HRV特异性序列，提示这些患者中存在HRV / HEV合并感染。该测定法已成功用于实验室工作人员同时发生的HRV呼吸系统疾病的调查。

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