A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA

Background: Enteroviruses (EVs) are significant human pathogens. Rapid and sensitive diagnostic techniques are desirable. Objectives: To develop a quantitative single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) for human enterovirus ribonucleic acid (RNA) (QPCR), with protection against amplimer contamination. Study design: The method was evaluated with serial dilutions of EV, 62 cerebrospinal fluid (CSF) specimens from meningitis patients, and the third and fourth European Union Concerted Action Enterovirus Proficiency Panels. A commercial EV PCR test was run in parallel. Results: Optimisations included RNA extraction procedure, design and concentrations of primers and probes from the 5' non-coding region as well as recombinant Thermus thermophilus polymerase (rTth), Mn(OAc)(2) and thermolabile UNG concentrations. Of 62 CSF samples from cases of meningitis submitted for QPCR testing, 34 (76%) and 21 (47%) were positive by QPCR and a commercial EV RNA detection kat, respectively. The detection limit of QPCR was 0.001 TCID50/ml (50% tissue culture-infective dose per millilitre) for a coxsackievirus B2 preparation and <10 copies of a plasmid containing coxsackievirus 132 complementary deoxyribonucleic acid (cDNA). The relation between threshold cycle (C-t) and amount of virus was linear (r = 0.99) over a range of 10(-3) to 10(4) TCID50/ml of coxsackievirus B2. Conclusions: The QPCR method allows a large number of samples to be screened rapidly. Its sensitivity, simplicity, and reproducibility make it a suitable tool for the routine laboratory. (C) 2003 Elsevier B.V. All rights reserved.