Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with anovel single-tube real-time reverse transcription-PCR assay

A single-tube real-time reverse transcription-PCR (RT-PCR) assay for entero virus detection in cerebrospinal fluid (CSF) was developed based on a fluor ogenic probe and primers directed to highly conserved sequences in the 5' u ntranslated region of the enterovirus genome. Quantitative detection of ent erovirus genome was demonstrated in a linear range spanning at least 5 logs . Endpoint titration experiments revealed that the in-tube detection limit of the assay was 11.8 enterovirus genome equivalents (95% detection rate) c orresponding in our current extraction protocol to 592 enterovirus genome e quivalents per ml of CSF. Twenty CSF specimens not suspected of viral menin gitis were all found to be negative, and no cross-reactivity with herpes si mplex virus type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza viruses A and B was observed. Nineteen CSF specimens from 70 patients suspected of viral meningitis were determined to be positive by PC R (27.1%), whereas only 17 were found to be positive by viral culture (24.3 %). The sensitivity of the assay was 100% and the specificity was 96.2% com pared to viral culture. Data from the real-time RT-PCR assay were available within 4 h. Our data suggest that the novel real-time RT-PCR assay may off er a reliable but significantly faster alternative to viral culture. Owing to the elimination of postamplification detection steps, its conduct requir ed considerably less hands-on time and was associated with a substantially reduced carryover risk compared to previously described PCR-based enterovir us detection assays.