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. 2018 Oct 30;15(1):167.
doi: 10.1186/s12985-018-1061-0.

A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus

Zhi-Shan Feng  1 , Li Zhao  2   3 , Ji Wang  3 , Fang-Zhou Qiu  2   3 , Meng-Chuan Zhao  1 , Le Wang  1 , Su-Xia Duan  1 , Rui-Qing Zhang  2   3 , Chen Chen  3 , Ju-Ju Qi  2   3 , Tao Fan  2   3 , Gui-Xia Li  4 , Xue-Jun Ma  5
Affiliations

A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus

Zhi-Shan Feng et al. Virol J. .

Abstract

Background: 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.

Results: 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.

Conclusion: mOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.

Keywords: Detection; HMPV; HRV; LNA; Multiplex one-tube nested real-time RT-PCR; RSV.

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Conflict of interest statement

Ethics approval and consent to participate

All aspects of the study were performed in accordance with national ethics regulations and approved by the Institutional Review Boards of National Institute for Viral Disease Control and Prevention, Center for Disease Control and Prevention of China.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
The sensitivity analysis and standard curves of the mOTNRT-PCR assay. a, b, and c are the amplification curves of RSV, HRV and HMPV, respectively using serial 10-fold dilutions of the mixed recombinant plasmids from 108 to 100 copies/μL, and d is the standard curves of the mOTNRT-PCR assay. The R2 of RSV, HRV and HMPV were 0.997, 0.994, 0.995 and the E of RSV, HRV and HMPV were 98.5, 95.7 and 92.5%, respectively
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