Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction for the detection of human enterovirus 71 subgenotype C4 in nasopharyngeal swab specimens

PLoS One. 2012;7(12):e52486. doi: 10.1371/journal.pone.0052486. Epub 2012 Dec 18.

Abstract

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID(50)) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Child
  • Child, Preschool
  • Enterovirus A, Human / genetics*
  • Enterovirus A, Human / isolation & purification
  • Hand, Foot and Mouth Disease / diagnosis*
  • Humans
  • Infant
  • Infant, Newborn
  • Nasopharynx / virology*
  • Nucleic Acid Amplification Techniques*
  • RNA, Viral
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction
  • Reverse Transcription*
  • Sensitivity and Specificity

Substances

  • RNA, Viral

Grant support

This work was supported by the China Mega-Project for Infectious Disease (2011ZX10004-001, 2012ZX10004-215) and a research grant from the State Key Laboratory for Genetic Engineering and Molecular Virology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.