Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4
Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4
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DOI：
10.1128/jcm.42.4.1564-1569.2004
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发表时间：
2004-04-01
影响因子：
9.4
通讯作者：
Claas, ECJ
中科院分区：
文献类型：
--
作者：
Templeton, KE;Scheltinga, SA;Claas, ECJ
病毒性呼吸道感染的实验室诊断通常通过细胞培养中的病毒分离和免疫荧光测定来进行。逆转录酶 PCR 现在被认为是检测呼吸道 RNA 病毒的敏感且特异的替代方案。开发了一种快速实时多重 PCR 检测方法，用于在两管多重反应中检测甲型流感病毒和乙型流感病毒、人呼吸道合胞病毒 (RSV)、副流感病毒 1 (PIV1.)、PIV2、PIV3 和 PIV4，并使用分子信标来区分病原体。通过多重检测对一年内采集的总共 358 个呼吸道样本进行了分析。通过培养和实时 PCR 检测，这些样本中检测到的呼吸道病毒的发病率分别为 358 例中的 67 例（19%）和 358 例中的 87 例（24%）。培养检测出 3 种甲型流感病毒、2 种乙型流感病毒、57 种 RSV、2 种 PIV1 和 2 种 PIV3 感染。通过多重实时 PCR 检测到所有这些培养阳性样本以及另外 5 种甲型流感病毒、6 种 RSV、2 种 PIV1、1 种 PIV2、1 种 PIV3 和 3 种 PIV4 感染。实时 PCR 在临床样本中的应用提高了呼吸道病毒诊断的灵敏度。此外，可以在 6 小时内获得结果，这增加了临床相关性。因此，使用这种实时 PCR 检测将改善患者管理和感染控制。
Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza virus 1 (PIV1.), PIV2, PIV3, and PIV4 in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens. A total of 358 respiratory samples taken over a I-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19%) and 87 of 358 (24%) by culture and real-time PCR, respectively. Culture detected 3 influenza A virus, 2 influenza B virus, 57 RSV, 2 PIV1, and 2 PIV3 infections. All of these culture-positive samples and an additional 5 influenza A virus, 6 RSV, 2 PIV1, I PIV2, I PIV3, and 3 PIV4 infections were detected by the multiplex real-time PCR. The application of real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within 6 h, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.