title: Quantitative real-time RT-PCR assay for research studies on enterovirus infections in the central nervous system.
abstract: 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.
authors: Clermont Université, Université d'Auvergne, EA4843, BP 10448, F-63000 Clermont-Ferrand, France. romain.volle@u-clermont1.fr
authors: Romain
authors: R
authors: Volle
authors: Céline
authors: C
authors: Nourrisson
authors: Audrey
authors: A
authors: Mirand
authors: Christel
authors: C
authors: Regagnon
authors: Martine
authors: M
authors: Chambon
authors: Cécile
authors: C
authors: Henquell
authors: Jean-Luc
authors: JL
authors: Bailly
authors: Hélène
authors: H
authors: Peigue-Lafeuille
authors: Christine
authors: C
authors: Archimbaud
mesh_terms: D002555:Cerebrospinal Fluid / Q000821:virology; D004770:Enterovirus / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D008582:Meningitis, Aseptic / Q000175:diagnosis* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods* / Q000592:standards