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Rhinovirus identification by BglI digestion of picornavirus RT-PCR amplicons
N G Papadopoulos1, J Hunter, G Sanderson
1University Medicine, Southampton General Hospital, UK.
Abstract:
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.
Insights
A new method uses BglI enzyme digestion to differentiate rhinoviruses from other picornaviruses. This rapid technique accurately identifies rhinoviruses, crucial for understanding the common cold and asthma exacerbations.
Area of Science:
- Virology
- Molecular Biology
- Respiratory Medicine
Background:
- Rhinoviruses are the primary cause of the common cold and trigger most asthma exacerbations.
- Current sensitive methods like RT-PCR cannot reliably distinguish rhinoviruses from other picornaviruses.
- Accurate identification of rhinoviruses is essential for clinical and research purposes.
Purpose of the Study:
- To develop a rapid and specific method for differentiating rhinoviruses from other picornaviruses.
- To leverage unique restriction enzyme sites within rhinovirus genetic material for identification.
Main Methods:
- Analysis of restriction maps of sequenced rhinoviruses identified a conserved BglI restriction site.
- This site is located in the middle of the amplicon generated by the OL26-OL27 primer pair.
- BglI digestion of amplicons was performed on various rhinovirus and other picornavirus samples.
Main Results:
- Rhinovirus amplicons consistently yielded a single 190-bp band after BglI digestion.
- Other picornaviruses showed varied results, with only 6.6% producing a single band pattern.
- The method demonstrated 100% sensitivity and over 90% specificity in distinguishing rhinoviruses.
Conclusions:
- BglI digestion of OL26-OL27 amplicons provides a rapid and easy way to distinguish rhinoviruses.
- This technique significantly improves the ability to differentiate rhinoviruses from other picornaviruses.
- The method has important implications for the diagnosis and study of respiratory infections.