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.