Development of a real-time RT-PCR assay for detection of resistance to oseltamivir in influenza A pandemic (H1N1) 2009 virus using single nucleotide polymorphism probes

Resistance to oseltamivir in pandemic (H1N1) 2009 influenza A virus is linked to an amino acid change from histidine (H) to tyrosine (Y) at position 275 in the neuraminidase protein (NA). A real-time one step RT-PCR assay using single nucleotide polymorphism (SNP) probes was developed to detect this mutation in respiratory specimens. The limit of detection was 47.6 copies/reaction for wild-type H275 RNA and 52.9 copies/reaction for the mutant H275Y RNA. The assay did not cross-react with other respiratory pathogens. The clinical sensitivity and specificity of the assay was compared to the gold standard Sanger sequencing method using 25 sensitive, 15 resistant and 20 negative samples. The sensitivity and specificity was 88.0% and 100% respectively with the SOIV_Osel_SEN probe designed to detect the H275 allele and 100% for the SOIV_Osel_RES probe detecting the 275Y allele. The sensitivity of the assay using nine admixtures of sensitive and resistant alleles was 88.9% and 77.8% with the SOIV_Osel_SEN probe and SOIV_Osel_RES probe respectively. The presence of mixed sensitive and resistant alleles in patient samples and mixtures of in vitro RNA were detected reproducibly. This assay can be used for screening of original samples for oseltamivir resistance without the need for culture and phenotypic testing.