One-step multiplex RT-qPCR for the detection and subtyping of influenza A virus in swine in Brazil

•A multiplex RT-qPCR with TaqMan probes was developed for IAV subtyping.•LODs ranged from 5.09 × 101 to 5.09 × 103 copies/μL.•The IAV subtype was determined in 74.0% (62.9–82.7, CI 95%) of the clinical samples.•The most frequently detected subtype was H3N2, followed by H1N1 and H1N2.•Different IAV lineages, mixed infections and reassortant viruses were detected. Pandemic H1N1, human-like H1N2 and H3N2 influenza A (IAV) viruses are co-circulating in swine herds in Brazil. The genetic analysis of the Brazilian IAVs has shown that they are genetically distinct from viruses found in swine in other countries; therefore, an update of the diagnostic assays for IAV detection and subtyping is needed. This study describes the development and validation of a TaqMan based – one-step multiplex RT-qPCR to discriminate the hemagglutinin and neuraminidase genes of the three major IAV subtypes circulating in pigs in Brazil. The RT-qPCR assays presented 100% (95.7–100, CI 95%) of diagnostic sensitivity in the analysis of 85 IAVs, previously characterized by sequencing. The limits of detection ranged from 5.09 × 101 to 5.09 × 103 viral RNA copies/μL. For the analytical specificity, 73 pig samples collected during 2017 and 2018 were analyzed, resulting in the identification of the subtype in 74.0% (62.9–82.7, CI 95%) of samples. From these, 46.3% were H3N2, 33.3% were H1N1, 11.1% were H1N2 and 3.7% were HxN1. Mixed viral infections (3.7%) and reassortant viruses (1.9%) were also detected by the test. This multiplex RT-qPCR assay provides a fast and specific diagnostic tool for identification of different subtypes and lineages of IAV in pigs, contributing to the monitoring of influenza in swine.