DIRECT AND UNINTERRUPTED RNA AMPLIFICATION OF ENTEROVIRUSES WITH COLORIMETRIC MICROWELL DETECTION

Background: Enteroviruses (EV) cause a broad spectrum of human disease s, of which aseptic meningitis is among the most common and most clini cally vexing. While the clinical symptoms of meningitis caused by bact eria, fungi and viruses are similar, the diagnosis, therapy and outcom e of disease caused by these agents vary greatly. In order to appropri ately manage meningitis patients, rapid and reliable diagnosis of EV m eningitis impacts significantly on patient management. Objective: To d evelop a direct and uninterrupted RNA amplification of enteroviruses u sing rTth DNA polymerase. Study design: Purified coxsackievirus B6 RNA of various concentrations was amplified by rTth DNA polymerase-mediat ed 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 o f cerebrospinal fluid (CSF). Results: All EV serotypes tested were det ected successfully by this method at a sensitivity of 1 TCID50 with th e exception of echoviruses 1, 5, 22 and 23. Echovirus 5 was detected a t 10 TCID50, and echovirus 1 was detected at 100 TCID50. Echoviruses 2 2 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 obser ved. Results of 10 CSF tested with this system correlated well with ti ssue culture. Conclusions: We have developed an EV amplification assay which has several important advantages over previously reported metho ds. This assay employs rTth DNA polymerase which possesses both revers e transcriptase and DNA polymerase activities, simplifying RNA reverse transcription and DNA amplification to an uninterrupted reaction. Add itionally, potential carryover contamination and enhanced amplificatio n specificity is provided by substituting dUTP for dTTP and adding ura cil N-glycosylase (UNG) in the amplification reaction. Finally, the de tection of amplified product is via a colorimetric, microwell format p ermitting the use of readily available instrumentation.