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A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus.

ABSTRACT: Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explored. 264 archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex Seeplex™ RV detection kit (Seegene) and the novel RSV LNA assay. The LNA assay demonstrated a significantly higher sensitivity than Seeplex, improving overall detection rates from 24% (64/264) to 32% (84/264). Detection limits of 9.0×10(1) and 6.0×10(2)copies/mL were observed for RSV A and B, respectively. RSV A was detected in 53/84 (63%) cases, and 31/84 (37%) were positive for RSV B. This novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of RSV.

SUBMITTER: Do LA

PROVIDER: S-EPMC3405522 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus.

Do Lien Anh Ha LA van Doorn H Rogier HR Bryant Juliet E JE Nghiem My Ngoc MN Nguyen Van Vinh Chau VC Vo Cong Khanh CK Nguyen Minh Dung MD Tran Tinh Hien TH Farrar Jeremy J de Jong Menno D MD

Journal of virological methods 20111119 1

Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explore ...[more]

PMID: 22119628

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Project description:BackgroundBoth respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens causing respiratory tract infection (RTI) in the pediatric population. However, the clinical manifestations of RSV and hMPV infections are similar. Therefore, a reliable and rapid diagnostic tool is needed for diagnostic performance.MethodsIn order to optimize diagnosis efficiency of RTI, the aim of this study is to establish a rapid and advanced method for simultaneous detecting RSV and hMPV in nasopharyngeal aspirates specimens from patients. We designed a one-step triplex real-time RT-PCR (qRT-PCR) protocol using TaqMan probes for detecting RSV and hMPV. The plasmid clones containing RSV nucleoprotein gene and hMPV fusion gene were established as reference standards. We used virus culture supernatants from 86 known pediatric RTI patient to test the specificity and sensitivity of our assay. Then we used total 222 nasopharyngeal aspirates specimens from pediatric patients hospitalized with respiratory symptoms to evaluate our assay.ResultsOur one-step triplex qRT-PCR assay showed 100% sensitivity and specificity in testing RSV and hMPV in 86 known virus culture supernatants, with excellent linearity (R2 > 0.99) and reliable reproducibility (CV lower than 1.04%). This assay has a wide dynamic range 102-109copies/reaction (limit of detection; LOD = 100 copies/reaction). A total of 222 patients hospitalized with respiratory symptoms were enrolled for clinical evaluation. In these samples, our qRT-PCR assay detected 68 RSV positive and 18 hMPV positive cases. However, standard virus culture only detected 8 RSV positive cases and 0 hMPV cases. Based on this improved triplex qRT-PCR assay, we found that RSV infection was associated with severe inflammation by chest X-ray and occurrence of pneumonia which were not observed previously.ConclusionsIn summary, we have developed a highly specific and sensitive one-step triplex qRT-PCR assay to detect hMPV and RSV simultaneously. This assay offers a valuable tool for routine diagnosis.

Dataset Information

A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus.

Publications

A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus.

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