Quantitative Real-Time RT-PCR Assay for Research Studies on Enterovirus Infections in the Central Nervous System

J Virol Methods. 2012 Oct;185(1):142-8. doi: 10.1016/j.jviromet.2012.06.019. Epub 2012 Jul 2.


Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections.

Publication types

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

MeSH terms

  • Cerebrospinal Fluid / virology
  • Enterovirus / isolation & purification*
  • Enterovirus Infections / diagnosis*
  • Enterovirus Infections / virology
  • Humans
  • Meningitis, Aseptic / diagnosis*
  • Meningitis, Aseptic / virology
  • Molecular Diagnostic Techniques / methods*
  • Molecular Diagnostic Techniques / standards
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Viral Load / methods*
  • Viral Load / standards