Direct and uninterrupted RNA amplification of enteroviruses with colorimetric microwell detection

Abstract. Background: Enteroviruses (EV) cause a broad spectrum of human diseases, of which aseptic meningitis is among the most common and most clinically vexing. While the clinical symptoms of meningitis caused by bacteria, fungi and viruses are similar, the diagnosis, therapy and outcome of disease caused by these agents vary greatly. In order to appropriately manage meningitis patients, rapid and reliable diagnosis of EV meningitis impacts significantly on patient management. Objective: To develop a direct and uninterrupted RNA amplification of enteroviruses using rTth DNA polymerase. Study design: Purified coxsackievirus B6 RNA of various concentrations was amplified by rTth DNA polymerase-mediated amplification to determine analytic sensitivity. The specificity of the EV amplification was examined with a panel of nucleic acids from 36 EV serotypes, 15 non-EV pathogens and 10 coded clinical specimens of cerebrospinal fluid (CSF). Results: All EV serotypes tested were detected successfully by this method at a sensitivity of 1 TCID₅₀ with the exception of echoviruses 1, 5, 22 and 23. Echovirus 5 was detected at 10 TCID₅₀, and echovirus 1 was detected at 100 TCID₅₀. Echoviruses 22 and 23 were not detectable at 100 TCID₅₀. Cross-reactivity of EV RT-PCR assay with 15 known non-EV meningitis pathogens has not been observed. Results of 10 CSF tested with this system correlated well with tissue culture. Conclusions: We have developed an EV amplification assay which has several important advantages over previously reported methods. This assay employs rTth DNA polymerase which possesses both reverse transcriptase and DNA polymerase activities, simplifying RNA reverse transcription and DNA amplification to an uninterrupted reaction. Additionally, potential carryover contamination and enhanced amplification specificity is provided by substituting dUTP for dTTP and adding uracil N-glycosylase (UNG) in the amplification reaction. Finally, the detection of amplified product is via a colorimetric, microwell format permitting the use of readily available instrumentation.