Rhinovirus identification by BglI digestion of picornavirus RT-PCR amplicons

Rhinoviruses are the main cause of the common cold and precipitate the majo rity of asthma exacerbations. RT-PCR followed by internal probe hybridisati on or Southern blotting, or nested PCRs are currently the most sensitive me thods for their identification. However, none of the published techniques c an differentiate satisfactorily rhinoviruses from other picornaviruses. Exa mination of the restriction maps of sequenced rhinoviruses, revealed a high ly conserved BglI restriction site (GCCnnnnnGGC), located exactly in the mi ddle of the 380-bp amplicon generated with the OL26-OL27 primer pair, which has been used extensively in the past to identify picornaviruses. Such a s ite was either not present, or positioned differently in other picornavirus es of known sequence. It was, therefore, considered that digestion of rhino virus amplicons with this enzyme would result in two equal length fragments , generating a single 190-bp band in gel electrophoresis. In contrast, eith er one undigested 380-bp band or a double-band pattern would appear in ampl icons from other picornaviruses. To test this hypothesis, Bgl digestions of OL26-OL27 amplicons from cultured and wild-type rhinoviruses, whose identi ty was confirmed by acid lability, as well as from echo, polio and coxsacki e viruses were carried out. All rhinovirus samples were digested successful ly generating single bands. Among the other picornaviruses, only 6.6% prese nted a single band pattern, while the rest were as predicted from the model . With a sensitivity of 100% and a specificity over 90%, the method describ ed, which is rapid and remarkably easy to perform, can be used to distingui sh rhinoviruses from other picornaviruses to a considerable extent. (C) 199 9 Elsevier Science B.V. All rights reserved.