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,Ji Wang,Xue-Jun Ma,Gui-Xia Li,Su-Xia Duan,Chen Chen,Rui-Qing Zhang,Ju-Ju Qi,Li Zhao,Meng-Chuan Zhao,Le Wang,Fang-Zhou Qiu,Tao Fan

doi:10.1186/s12985-018-1061-0

Abstract

BackgroundRespiratory 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.ResultsA 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.ConclusionmOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.

Highlights

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
We adopted the design of locked nucleic acid (LNA)-modified primers and develop a multiplex one-tube nested real time RT-Polymerase chain reaction (PCR) assay for simultaneous detection of RSV, HRV and HMPV with the advantages of higher sensitivity, more convenience and cost-effectiveness than individual Traditional real time PCR (RT-qPCR)
Sensitivity and specificity for mOTNRT-PCR assay As shown in (Fig. 1a, b and c), the sensitivities of mOTNRT-PCR assay for RSV, HRV and HMPV were 5 copies /reaction

Summary

Introduction

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. Acute respiratory tract infections causes high morbidity and mortality in hospitalized patients and nearly 80% of infections are viral [1,2,3]. Respiratory syncytial virus (RSV), human rhinovirus (HRV) and human metapneumovirus (HMPV) are the main causative agents of acute respiratory tract infections in humans worldwide across. Multiplex PCR or traditional real time PCR (RT-qPCR) assays have been developed worldwide for the detection. We adopted the design of locked nucleic acid (LNA)-modified primers and develop a multiplex one-tube nested real time RT-PCR (mOTNRT-PCR) assay for simultaneous detection of RSV, HRV and HMPV with the advantages of higher sensitivity, more convenience and cost-effectiveness than individual RT-qPCR. No unspecific amplification or detection of mOTNRT-PCR was observed with these specimens (data not shown), suggesting the high specificity of mOTNRT-PCR assay

Methods

Results

Conclusion