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J Virol Methods. 2018 Aug;258:7-12. doi: 10.1016/j.jviromet.2018.05.005. Epub 2018 May 26.

Rapid detection of hand, foot and mouth disease enterovirus genotypes by multiplex PCR.

Author information

1
Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in Universities of Shandong, Taishan Medical College, Taian, 271000, Shandong, China.
2
Department of Children's Medical Laboratory Diagnosis Center, Qilu Chidren's Hospital of Shandong University, Jinan, Shandong, 250022, China.
3
Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo, 001-0020, Japan; National Virus Reference Laboratory, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
4
Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in Universities of Shandong, Taishan Medical College, Taian, 271000, Shandong, China. Electronic address: shiwf@ioz.ac.cn.

Abstract

Hand, foot and mouth disease (HFMD) is a pediatric disease associated with infection by enterovirus (EV) genotypes. The major HFMD EV pathogens are enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16); however, recently, coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) have also emerged. EV genotypes cannot be distinguished on clinical grounds and a new methodology for the rapid detection of the four major HFMD EV genotypes is urgently required. In the present study, a multiplex real-time PCR assay was established for the simultaneous detection of CVA6, CVA10, CVA16 and EVA71. The specificity and sensitivity of the assay was determined on a validation panel of clinical samples, comprising cerebrospinal fluid (n = 51), blood (n = 39), feces (n = 58) and throat swabs (n = 29). The results showed that the multiplex real-time PCR exhibited high specificity, no cross-reactivity with other EV genotypes, lower limits of detection for CVA6, CVA10, CVA16 and EVA71 were 4 × 103, 4 × 102, 5 × 102, and 3 × 103 copies/μL, respectively and had comparable sensitivity to singleplex assays testing clinical samples. The multiplex real-time PCR methodology established in this study can be employed for the rapid detection of the four most prevalent HFMD-associated EVs, for epidemiologic surveillance of circulating EV genotypes and for assessing treatment responses and vaccine studies.

KEYWORDS:

CVA10; CVA16; CVA6; EVA71; Enterovirus; HFMD; Multiplex PCR

PMID:
29758237
DOI:
10.1016/j.jviromet.2018.05.005
[Indexed for MEDLINE]

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