Rapid detection of enterovirus in clinical specimens using PCR and microwell capture hybridization assay

A rapid detection method of enteroviral RNA in clinical samples using PCR and a microwell capture hybridization assay is described. PCR products were labelled directly by digoxigenin-dUTP during the amplification step. The labelled amplicons were hybridized with a biotinylated oligo-probe and captured on commercially available test microwells coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and colorimetric reaction automatically measured. This method detected as few as 0.01 PFU/100 microl of biological sample with a result obtained within 8 h. Using this method, we were able to detect enteroviral RNA in 23 of 35 clinical specimens from 16 of 17 patients with suspected acute or chronic enteroviral infection. The samples included cerebrospinal fluid, broncho-pulmonary lavage, pericardial effusion, throat swabs, stools, sera, muscular and myocardial biopsies. In contrast, virus was isolated in cell culture in only 8 of 28 clinical specimens from 6 of the 17 patients. This easy-to-perform assay has useful potential in the rapid detection of enterovirus in acute or chronic infection. This methodology could be used for a rapid qualitative detection of other RNA viruses.