Rapid Detection of the H275Y Oseltamivir Resistance Mutation in Influenza A/H1N1 2009 by Single Base Pair RT-PCR and High-Resolution Melting

摘要: Introduction: We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses. Findings: A novel strategy of amplifying single base pair, relevant SNP at position 823 neuraminidase gene, was chosen maintain specificity assay. Wildtype and mutant virus were differentiated when using known reference samples cell-cultured virus. However, dilutions these assayed, amplification nonspecific primer-dimer evident affected overall temperature (Tm) amplified products. Due appearance .30 cycles we found if cycle threshold (CT) for dilution .30, HRM did not consistently discriminate from wildtype. Where CT ,30 noted an inverse relationship between Tm fitted quadratic curves allowed discrimination wildtype, 30:70 mutant:wildtype mixtures. compared values TaqMan H1N1 09 detection with those 59 clinical demonstrated CT.32.98 would have CT.30. Analysis 609 consecutive predicted 207 (34%) result CT.30 therefore be amenable Conclusions: The use pair PCR can useful specifically interrogating SNPs. When applied 09, constraints this placed on primer resulted impact had adjusted by plotting against CT. Although less sensitive than assays, rapidly, low cost, screen moderate viral concentrations.