Use of heat labile UNG in an RT-PCR assay for enterovirus detection

A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to replace the Roche AMPLICOR Enterovirus Test used in our laboratory from 1996 to 1999. The new assay design was optimized to match or exceed the performance of the Roche AMPLICOR Enterovirus test kit with respect to analytical sensitivity and specificity, contamination control, ease of use and availability of reagents. This new assay uses a heat labile form of the enzyme uracil DNA glycosylase (UNG) for amplicon contamination control and an RT-PCR enzyme mixture, enabling a one tube/one step amplification. RNA preparation was undertaken using a commercial extraction kit. End detection was accomplished using a probe-capture enzyme immuno assay (EIA) plate format. This EV RT-PCR assay exceeds the performance of the Roche AMPLICOR Enterovirus assay in a direct comparison. The combined enzymological approach has potential application to a wide variety of assays requiring sensitive RNA detection and stringent contamination control, including those utilizing real time detection methods.