A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus.
A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus.
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DOI：
10.1186/s12985-018-1061-0
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发表时间：
2018-10-30
期刊：
影响因子：
4.8
通讯作者：
Ma XJ
中科院分区：
文献类型：
--
作者：
Feng ZS;Zhao L;Wang J;Qiu FZ;Zhao MC;Wang L;Duan SX;Zhang RQ;Chen C;Qi JJ;Fan T;Li GX;Ma XJ
呼吸道合胞病毒（RSV）、人鼻病毒（HRV）和人间质肺病毒（HMPV）是引起住院患者急性呼吸道感染的重要病毒病原体。灵敏、准确地检测RSV、HRV和HMPV对于临床诊断和治疗是必要的。开发了一种基于锁核酸 (LNA) 的多重封闭单管巢式实时 RT-PCR (mOTNRT-PCR) 检测方法，用于同时检测 RSV、HRV 和 HMPV。对 mOTNRT-PCR 的敏感性、特异性、重现性和临床表现进行了评估，并与使用临床样本的个体实时 PCR (RT-qPCR) 检测进行比较。 mOTNRT-PCR 检测对 RSV、HRV 和 HMPV 的分析灵敏度分别为 5 个拷贝/反应，未观察到与其他常见呼吸道病毒的交叉反应。测定内和测定间的变异系数（CV）在0.51%至3.67%之间。在检测的 398 份鼻咽吸出物样本中，RSV、HRV 和 HMPV 阳性分别为 109 份（27.39%）、150 份（37.69%）和 44 份（11.06%），而 RSV、HRV 和 HMPV 阳性分别为 95 份（23.87%）、137 份（34.42%）和 38 份（9.55%）。分别通过单独的 RT-qPCR 检测。使用报道的传统两步巢式 PCR 测定进行测序，确认 mOTNRT-PCR 呈阳性但 RT-qPCR 呈阴性的 33 个样本为真阳性。 mOTNRT-PCR 检测在临床环境中检测 RSV、HRV 和 HMPV 时显示出比 RT-qPCR 检测更高的灵敏度。本文的在线版本 (10.1186/s12985-018-1061-0) 包含补充材料，可供授权用户使用。
Respiratory syncytial virus (RSV), human Rhinovirus (HRV) and human Metapneumo Virus (HMPV) are important viral pathogens causing acute respiratory tract infections in the hospitalized patients. Sensitive and accurate detection of RSV, HRV and HMPV is necessary for clinical diagnosis and treatment. A locked nucleic acid (LNA)-based multiplex closed one-tube nested real-time RT-PCR (mOTNRT-PCR) assay was developed for simultaneous detection of RSV, HRV and HMPV. The sensitivity, specificity, reproducibility and clinical performance of mOTNRT-PCR were evaluated and compared with individual real time PCR (RT-qPCR) assay using clinical samples. The analytical sensitivity of mOTNRT-PCR assay was 5 copies/reaction for RSV, HRV and HMPV, respectively, and no cross-reaction with other common respiratory viruses was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.51 to 3.67%. Of 398 nasopharyngeal aspirates samples tested, 109 (27.39%), 150 (37.69%) and 44 (11.06%) were positive for RSV, HRV and HMPV, respectively, whereas 95 (23.87%), 137 (34.42%) and 38 (9.55%) were positive for RSV, HRV and HMPV, respectively, by individual RT-qPCR assay. Thirty three samples that were positive by mOTNRT-PCR but negative by RT-qPCR were confirmed as true positives by sequencing using reported traditional two-step nested PCR assay. mOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings. The online version of this article (10.1186/s12985-018-1061-0) contains supplementary material, which is available to authorized users.
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DOI：
10.1016/j.diagmicrobio.2017.03.010
发表时间：
2017-06
影响因子：
2.9
作者：
Zhao MC;Li GX;Zhang D;Zhou HY;Wang H;Yang S;Wang L;Feng ZS;Ma XJ
通讯作者：
Ma XJ
影响因子：
3.1
作者：
Bellau-Pujol S;Vabret A;Legrand L;Dina J;Gouarin S;Petitjean-Lecherbonnier J;Pozzetto B;Ginevra C;Freymuth F
通讯作者：
Freymuth F
影响因子：
12.7
作者：
Sanghavi, Sonali K.;Bullotta, Arlene;Rinaldo, Charles R.
通讯作者：
Rinaldo, Charles R.
影响因子：
9.4
作者：
Hu, AZ;Colella, M;Cheng, SM
通讯作者：
Cheng, SM
影响因子：
9.4
作者：
Maertzdorf, J;Wang, CK;Fouchier, RAM
通讯作者：
Fouchier, RAM