SCREENING OF H275Y MUTATION IN INFLUENZA A (H1N1) pdm09 ISOLATED IN MALAYSIA BY RAPID REAL-TIME PCR ASSAYS

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J. S. K. A. O. R. T. F. M. K. Mohd. Apandi Yusof, T.S. Saraswathy, Zainah Saat, “SCREENING OF H275Y MUTATION IN INFLUENZA A (H1N1) pdm09 ISOLATED IN MALAYSIA BY RAPID REAL-TIME PCR ASSAYS”, ijmhs, vol. 3, no. 5, Sep. 2013.
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Abstract

During the influenza A(H1N1) pdm09 outbreak, there were concerns about drug resistance to oseltamivir due to a mutation in the influenza A(H1N1) virus. The mutation at amino acid position 275 resulted from a substitution of histidine to tyrosine (H275Y) at the neuraminidase (N1) region was responsible to confer resistance to aseltamivir. The existing methods of detecting resistant viruses by sequencing and NA inhibition assays are laborious and time consuming. In this study, 120 influenza A(H1N1) pdm09 Malaysian viral isolates collected during the 2009-2010 pandemic season were tested for the presence of H275Y mutation by Taqman Dual Probe Real-Time PCR and High Resolution Melting (HRM) assays. The Taqman Real-Time PCR and HRM assays detect and distinguish wild type and mutant H275Y virus using dual probe and a saturation dye respectively. The difference in the sigmoidal curve in the Taqman Assay and the shift in the melt curve in HRM assay occurred when a single nucleotide variation is detected. All 120 influenza A(H1N1) pdm09 Malaysian  isolates were found to be wild type. This finding was consistent with sequencing results. However, the developed HRM assay was found to be robust and cheaper than sequencing and Taqman assay.

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