Rhinovirus identification by BglI digestion of picornavirus RT-PCR amplicons.
Rhinoviruses are the main cause of the common cold and precipitate the majority of asthma exacerbations. RT-PCR followed by internal probe hybridisation or Southern blotting, or nested PCRs are currently the most sensitive methods for their identification. However, none of the published techniques can differentiate satisfactorily rhinoviruses from other picornaviruses. Examination of the restriction maps of sequenced rhinoviruses, revealed a highly conserved BglI restriction site (GCCnnnnnGGC), located exactly in the middle 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 site was either not present, or positioned differently in other picornaviruses of known sequence. It was, therefore, considered that digestion of rhinovirus amplicons with this enzyme would result in two equal length fragments, generating a single 190-bp band in gel electrophoresis. In contrast, either one undigested 380-bp band or a double-band pattern would appear in amplicons from other picornaviruses. To test this hypothesis, Bgl digestions of OL26-OL27 amplicons from cultured and wild-type rhinoviruses, whose identity was confirmed by acid lability, as well as from echo, polio and coxsackie viruses were carried out. All rhinovirus samples were digested successfully generating single bands. Among the other picornaviruses, only 6.6% presented 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 described, which is rapid and remarkably easy to perform, can be used to distinguish rhinoviruses from other picornaviruses to a considerable extent.
Papadopoulos
N G
NG
University Medicine, Southampton General Hospital, UK.
Hunter
J
J
Sanderson
G
G
Meyer
J
J
Johnston
S L
SL
D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D010849:Picornaviridae / Q000235:genetics; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012229:Rhinovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D015252:Deoxyribonucleases, Type II Site-Specific; C018492:GCCNNNNNGGC-specific type II deoxyribonucleases