A one-step, triplex, real-time RT-PCR assay for the simultaneous detection of enterovirus 71, coxsackie A16 and pan-enterovirus in a single tube

PLoS One. 2014 Jul 16;9(7):e102724. doi: 10.1371/journal.pone.0102724. eCollection 2014.

Abstract

The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • China
  • DNA Primers / genetics
  • Enterovirus / isolation & purification*
  • Enterovirus A, Human / isolation & purification*
  • Hand, Foot and Mouth Disease / diagnosis*
  • Hand, Foot and Mouth Disease / virology*
  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Real-Time Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*

Substances

  • DNA Primers

Grants and funding

This study was supported by the Natural Science Foundation of the Fujian Province (2013J06016) and National Major Scientific and Technological Special Project for Significant New Drug Development (2011ZX2009401-403-16). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.