- Pyrosequencing
Pyrosequencing is a molecular technique that can be used for the detection and quantitation of neuraminidase inhibitor resistance mutations. This rapid technique can be used on cultured virus and directly on clinical material which means that it can be used to inform individual patient management. It has the advantage over current methodologies which are more time consuming or lack the sensitivity to detect mutations in mixed virus populations.
Pyrosequencing is a real-time DNA sequencing technique which, via a cascade of enzymatic reactions, detects pyrophosphate (PPi) released during DNA synthesis as visible light. The light released is quantitative and enables the rapid generation of sequence information.
This rapid technique is not yet in widespread use due to some limitations: the equipment and reagents are expensive, with the instrumentation costing in the region of £60,000, and if used alone it can only be used to detect well-characterised mutations. Nonetheless, the technique is suitable for high throughput surveillance or drug resistance screening, as demonstrated during the emergence of the oseltamivir resistant seasonal influenza A (H1N1) H274Y viruses in Europe in 2007-08.
Pyrosequencing protocols
Protocol for pandemic influenza A N1 (H1) gene for NAI sensitivity
The protocol describes the method for amplification and pyrosequencing of the 2009 pandemic influenza (H1) N1 gene to detect the H275Y mutation, which causes resistance to the neuraminidase inhibitor oseltamivir.
Pyrosequencing protocol (provided by kind permission of the Health Protection Agency, London)
Protocol for seasonal influenza A N1 (H1) gene for NAI sensitivity
The protocol describes the method used to amplify the N1 gene from nucleotide 601 to 931. This region allows pyrosequencing of the mutation encoding the H274Y change reported to cause resistance to the neuraminidase inhibitor oseltamivir.
Pyrosequencing protocol (provided by kind permission of the Health Protection Agency, London)
Protocols for pandemic influenza NAI sensitivity
The WHO has published protocols to amplify the M2 gene and NA-274 of the pandemic influenza A (H1N1) virus. These are available on the WHO website.
Film of pyrosequencing methodology
The short film explains pyrosequencing methodology and demonstrates how the technique is undertaken.
Pyrosequencing film here
Provided by kind permission of the Health Protection Agency, London.
Pyrosequencing publications
- Lackenby A, Democratis J, Siqueira MM, Zambon MC (20088). Rapid quantitation of neuraminidase inhibitor drug resistance in influenza virus quasispecies. Antivir Ther. 2008;13(6):809-20.
- Deyde VM, Okomo-Adhiambo M. Sheu TG, Wallis TR, Fry A, Dharan N, Klimov AI, Gubareva LV. (2008). Pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza A viruses. Antivir Res 2009; 81(1):16-24.
- Duwe S, Schweiger B (2008). A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1. J Virol Methods. 2008 Nov;153(2):134-41. Epub 2008 Sep 19.
