Development and clinical testing of a simple, low-density gel element array for influenza identification, subtyping,...

The objectives of this study were to develop a user-friendly, gel element microarray test for influenza virus detection, subtyping, and neuraminidase inhibitor resistance detection, assess the performance characteristics of the assay, and perform a clinical evaluation on retrospective nasopharyngeal swab specimens. A streamlined microarray workflow enabled a single user to run up to 24 tests in an 8h shift. The most sensitive components of the test were the primers and probes targeting the A/H1 pdm09 HA gene with an analytical limit of detection (LoD) <100 gene copies (gc) per reaction. LoDs for all targets in nasopharyngeal swab samples were ≤1000 gc, with the exception of one target in the seasonal A/H1N1 subtype. Seasonal H275Y variants were detectable in a mixed population when present at >5% with wild type virus, while the 2009 pandemic H1N1 H275Y variant was detectable at ≤1% in a mixture with pandemic wild type virus. Influenza typing and subtyping results concurred with those obtained with real-time RT-PCR assays on more than 97% of the samples tested. The results demonstrate that a large panel of single-plex, real-time RT-PCR tests can be translated to an easy-to-use, sensitive, and specific microarray test for potential diagnostic use.