Rapid molecular detection of the H275Y oseltamivir resistance gene mutation in circulating influenza A (H1N1) viruses

Abstract

In early 2008, drug susceptibility surveillance of influenza viruses in Europe revealed that some influenza A viruses (subtype H1N1) circulating during the winter season of 2007 and 2008 were resistant to the neuraminidase inhibitor, oseltamivir. This resistance arises due to a histidine to tyrosine substitution in the neuraminidase active site (H275Y in N1 nomenclature). Current methods to detect this mutation involve an end-point reverse transcription polymerase chain reaction followed by nucleotide sequencing. While accurate, this approach has the limitation of being time-consuming, labour-intensive and expensive. Herein we describe a one-step allelic discrimination assay which rapidly (2h) detects this resistance mutation. The sensitivity of the assay was as low as 10 copies per reaction and is capable of detecting the antiviral resistance mutation in a mixture of wild type H275 and mutant H275Y targets.

Fig. 1

Determination of the limit of detection of the AD RT-PCR assay for influenza A/N1 H275 (A) and H275Y (B). Quantified standards in the range 10⁸ to 10¹ copies were run in triplicate for each target and plotted as Ct (crossing threshold) values versus the log of the copy number of the standard. PCR parameters are included for each assay.

Fig. 2

Detection of oseltamivir resistant influenza A/H1N1 H275Y from clinical samples by allelic discrimination real-time RT-PCR. Delta Rn versus real-time cycle number plots showing fluorescent amplification curves from wild-type influenza A/H1N1 (H275, oseltamivir sensitive) with the VIC-TaqMan probe (A) and from A/H1N1 H275Y (B) oseltamivir resistant mutants with the FAM-TaqMan probe. Rn is the normalised signal reporter fluorescence divided by the passive internal reference dye, ROX.

Fig. 3

Component Plots of fluorescence emission versus cycle number of mixed populations of influenza A/N1 H275 and H275Y amplicons. Detection of 500 copies of the influenza A/N1 H275 oseltamivir sensitive amplicon and 500 copies of an influenza A/N1 H275Y oseltamivir resistant amplicon by AD RT-PCR showing an increase in both FAM fluorescence and VIC fluorescence (A); 900 copies of an oseltamivir sensitive influenza A/N1 H275 amplicon mixed with 100 copies of an oseltamivir resistant influenza A/N1 H275Y amplicon was also detectable by the AD RT-PCR approach showing an increase in both FAM and VIC fluorescence (B).