Solution hybridization and enzyme immunoassay for biotinylated DNA-RNA hybrids to detect enteroviral RNA in cell culture

Abstract

A non-isotopic hybridization assay is described for detection of enteroviral RNA in cell culture. Two biotin-labelled cDNA probes, corresponding to 1 kb from the 5′ end and 3·5 kb from the 3′ end of the coxsackievirus B3 genome, were hybridized in solution with protease and detergent-treated cell culture suspensions. Labelled DNA-RNA hybrids were captured on microtiter plates coated with anti-biotin antibody and bound hybrids were measured with a β-galactosidase-labelled monoclonal antibody specific for DNA-RNA hybrids. Coxsackie B3 was detected at a concentration of 500 pfu ml −1. The limit of detection for other enteroviruses ranged from 10 3·3 to 10 5·8 pfu ml −1. The enteroviruses that could be detected included coxsackie B1 and 3, coxsackie A1-6 and 15, poliovirus types 1–3, and enteroviruses 7, 11, and 71. ECHO 22 was the only enterovirus, of those that were tested, that could not be detected. The solution hybridization reaction and enzyme immunoassay for DNA-RNA hybrids does not require the use of radiolabelled probes or extraction of RNA with phenol. The assay yields a quantitative endpoint, which avoids the subjectivity inherent in membrane-based methods. These features would make the assay more adaptable to clinical laboratories than other formats which have been devised for measurement of viral RNA.