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Open Access Highly Accessed Methodology

Rapid and sensitive identification of RNA from the emerging pathogen, coxsackievirus A6

Lei Zhang1, Xinying Wang1, Yanjun Zhang1*, Liming Gong1, Haiyan Mao1, Cen Feng1, David M Ojcius4 and Jie Yan23*

Author Affiliations

1 Zhejiang Provincial Center for Disease Control and Prevention, 630 Xincheng Road, Hangzhou, Zhejiang, 310051, P.R. China

2 Division of Basic Medical Microbiology, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310003, P.R. China

3 Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, 866 Yu-Hang-Tang Road, Hangzhou, Zhejiang, 310058, P.R. China

4 Health Sciences Research Institute and School of Natural Sciences, University of California, Merced, CA, 95343, USA

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Virology Journal 2012, 9:298  doi:10.1186/1743-422X-9-298

Published: 30 November 2012

Abstract

Background

Hand, foot and mouth disease (HFMD) is caused by members of the family Picornaviridae in the genus Enterovirus. It has been reported that coxsackievirus A6 (CVA6) infections are emerging as a new and major cause of epidemic HFMD. Sporadic HFMD cases positive for CVA6 were detected in the mainland of China in recent years. To strengthen the surveillance of CVA6 infections and outbreak control, the clinical diagnosis is urgently needed to distinguish the CVA6 infection disease from other infections.

Methods

In order to develop a sensitive quantitative real-time RT-PCR assay for rapid detection of CVA6 RNA, primers and probe were designed to target the VP1 gene segment of CVA6. The conservation of the target segment was firstly analyzed by bioinformatic technology. The specificity of the real-time RT-PCR was further confirmed by detecting other related viruses and standard curves were established for the sensitivity evaluation. The pharyngeal swab samples from the EV71 and CVA16 unrelated HFMD patients were applied for CVA6 detection through the established method.

Results

Based on the primer–probe set to detect the target VP1 gene segment of CVA6, the quantitative real-time RT-PCR assay could discriminate CVA6 infection from other resemble viral diseases with a potential detection limit of 10 viral copies/ml. The specificity of the assay was determined by sequence alignment and experimentally tested on various related viruses. The standard curve showed that the amplification efficiency of templates with different concentrations of templates was almost the same (R2 >0.99). Evaluation of the established method with pharyngeal swabs samples showed good accordance with the results from serology diagnosis.

Conclusion

This study is the first report developing a VP1 gene-based quantitative real-time RT-PCR for rapid, stable and specific detection of CVA6 virus. The real-time RT-PCR established in this study can be used as a reliable method for early diagnosis of CVA6 infection.

Keywords:
Hand; Foot and mouth disease; Enterovirus; Coxsackievirus A6; Quantitative real-time RT-PCR