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  • Title: Detection of influenza A H1N1 and H3N2 mutations conferring resistance to oseltamivir using rolling circle amplification.
    Author: Steain MC, Dwyer DE, Hurt AC, Kol C, Saksena NK, Cunningham AL, Wang B.
    Journal: Antiviral Res; 2009 Dec; 84(3):242-8. PubMed ID: 19800370.
    Abstract:
    In the event of an influenza pandemic, the use of oseltamivir (OTV) will undoubtedly increase and therefore it is more likely that OTV-resistant influenza strains will also arise. OTV-resistance genotyping using sequence-based testing on viruses isolated in cell culture is time consuming and less likely to detect the low-level presence of drug-resistant virus populations. We have developed a novel rolling circle amplification (RCA) method to achieve the sensitive detection of OTV-resistant viruses from clinical specimens. Using artificially created templates, RCA could detect the presence of OTV-resistant mutations (N2: 119V, 292K, N1: 274Y) even if the population carrying the mutations was <1% of the total. By applying RCA to clinical samples, we identified the emergence of the 274Y mutation in one OTV-treated patient, as well as in seven individuals who were treatment-naïve (confirming community transmission of 274Y-containing resistant influenza A H1N1). These results were further confirmed by neuraminidase region sequencing. In conclusion, RCA technology can provide rapid (<24 h), high-throughput diagnosis of OTV resistance mutations with a high specificity and sensitivity.
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