A six‐plex droplet digital RT‐PCR assay for seasonal influenza virus typing, subtyping, and lineage determination

BACKGROUND: There are two influenza A subtypes (H1 and H3) and two influenza B lineages (Victoria and Yamagata) that currently co‐circulate in humans. In this study, we report the development of a six‐plex droplet digital RT‐PCR (ddRT‐PCR) assay that can detect HA and M segments of influenza A (H1, H3, and M) and influenza B (Yamagata HA, Victoria HA, and M) viruses in a single reaction mixture. It can simultaneously detect six different nucleic acid targets in a ddRT‐PCR platform. METHODS: The six‐plex ddRT‐PCR used in this study is an amplitude‐based multiplex assay. The analytical performance of the assay was evaluated. Correlation with standard qRT‐PCR methodology was assessed using 55 clinical samples. RESULTS: The assay has a wide dynamic range, and it has good reproducibility within and between runs. The limit of quantification of each target in this assay ranged from 15 copies/reaction for influenza B Victoria M gene to 45 copies/reaction for influenza B Yamagata M gene. In addition, this assay can accurately quantify each of these targets in samples containing viral RNAs from two different viruses that were mixed in a highly skewed ratio. Typing, subtyping, and lineage differentiation data of 55 tested clinical respiratory specimens were found to be identical to those deduced from standard monoplex qRT‐PCR assays. CONCLUSIONS: The six‐plex ddRT‐PCR test was demonstrated to be highly suitable for detecting dual influenza infection cases. This assay is expected to be a useful diagnostic tool for clinical and research use.