Diagnostic accuracy of an allele-specific reverse transcriptase-PCR assay targeting the H275Y oseltamivir resistant mutation in 2009 pandemic influenza A/H1N1 virus.

摘要: Abstact Background Oseltamivir resistant 2009 pandemic influenza A/H1N1 viruses (pH1N1) are emerging and rapid molecular assays identifying these strains needed for clinical management. Objective Development evaluation of an allele-specific, real-time reverse transcriptase-PCR assay (ASPCR) targeting the H275Y oseltamivir mutation in pH1N1 virus. Study design ASPCR uses two allele-specific forward primers (wild-type mutant) a common primer probe. Wild-type mutant genotypes were defined by difference PCR Ct values (ΔCtmut–wt) between wild-type amplification curves same sample. Mixtures analyzed to evaluate sensitivity determine cut-off values. results confirmed using allelic discrimination (AD) pyrosequencing. Results containing 5–95% genotype could be detected. A ΔCtmut–wt ≥ 3.5 identified ( 90% mutant). Among 264 samples, 171 wild-type, 10 mixed, 29 fully mutant. The 39 samples with mixed or from 11 patients. Of 107 sufficient volume tested AD, 12 indeterminate AD due low viral load, 86 both assays, 9 assays. Thirteen pyrosequencing one discrepant sample was Conclusions is sensitive, quantitative specific analysis provides accurate approach detecting resistance samples.