Single tube real-time RT-PCR Assay for the detection of enterovirus

We developed a rapid and sensitive method for the routine detection of enterovirus in CSF and plasma using a one tube, one step, real-time PCR assay. A primer set and an Eclipse probe were designed to target the highly conserved 5' untranslated region of the enterovirus genome. This assay was compared to a commercial PCR-ELISA assay. In an initial clinical validation, the newly developed real-time PCR assay provided 92% sensitivity, 100% specificity, 100% positive predictive value, and 97% negative predictive value compared to the commercial assay. During test validation, we detected 2 single nucleotide polymorphisms beneath the Eclipse probe sequence. To increase clinical sensitivity, we designed a modified degenerate Eclipse probe using neutral and specialized "super" bases from Epoch Biosciences (Bothell, WA). This modified probe assay demonstrated 100% agreement with the PCR-ELISA assay. The existence of unforeseen polymorphisms in genomes targeted by probes in realtime PCR assays can confound detection. Modified bases are useful tools for accommodating sequence variations in real-time assays.