Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR
Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR
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DOI：
10.1016/j.jcv.2004.08.021
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发表时间：
2005-08-01
影响因子：
8.8
通讯作者：
Claas, ECJ
中科院分区：
文献类型：
--
作者：
Scheltinga, SA;Templeton, KE;Claas, ECJ
背景资料：人鼻病毒（hRV）和人偏肺病毒（hMPV）的充分实验室诊断需要分子方法，因为病毒培养缺乏敏感性。然而，对所有呼吸道病毒建立单独的PCR是不切实际的，因此优先使用多重PCR。目的：开发一种用于常规诊断的快速实时PCR检测方法，用于检测hRV和hMPV，包括在单管多重反应中使用携带不同荧光团的探针来区分靶标的内部对照。优化多重实时RNA PCR以包括内部对照病毒，并通过多重测定分析了一年内从239名患者采集的总共358份呼吸道样本。与内部对照共扩增的多重测定与单独测定一样灵敏和特异。在一年时间内对239例患者的临床样本应用该检测，hRV和hMPV的发生率分别为41/239（17.1%）和6/239（2.5%）。在8份（2.2%）样本中检测到抑制（定义为内部对照扩增不良）。文化进行这些样品，只有四个hRV detected.Conclusions：这种实时PCR方法，使这两个呼吸道病原体的敏感诊断的潜力，扩大检测的一部分，一个完整的分子呼吸道病毒屏幕。(C)2005 Elsevier B. V.保留所有权利。
Background: Adequate laboratory diagnosis of human rhinoviruses (hRV) and human metapneumoviruses (hMPV) requires molecular methods as viral culture lacks sensitivity. However, setting up individual PCRs for all respiratory viruses is not practical so preferentially multiplex PCRs are used.Objectives: To develop for routine diagnosis a rapid real-time PCR assay for detection of hRV and hMPV including an internal control in a single tube multiplex reaction using probes carrying different fluorophores to discriminate targets.Study design: The multiplex real-time RNA PCR was optimized to include the internal control virus and a total of 358 respiratory samples from 239 patients taken over a one-year period were analyzed by the multiplex assay.Results: The multiplex assay with co-amplification of the internal control was as sensitive and specific as the individual assays. Application of this assay on clinical samples from 239 patients in a one-year period resulted in an incidence of hRV and hMPV of 41/239 (17.1%) and 6/239 (2.5%), respectively. Inhibition, defined as poor internal control amplification, was detected in 8 (2.2%) samples. Culture was performed on these samples and only four hRV were detected.Conclusions: This real-time PCR method enables sensitive diagnosis of these two respiratory pathogens with the potential to expand the assay as part of a full molecular respiratory viral screen. (C) 2005 Elsevier B.V. All rights reserved.