A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses

Virological and serological surveys have documented that H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 influenza viruses are consistently circulating in dogs. In the present study, a multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection and differentiation of these influenza viruses. Four primer sets were designed to target the hemagglutinin genes of H1N1/2009, cH3N2, hH3N2, and H3N8 canine influenza viruses (CIVs). This mRT-PCR assay demonstrated high specificity and sensitivity for the four CIV subtypes. Additionally, mRT-PCR results obtained from 420 clinical samples were consistent with those obtained by the conventional virus isolation method. Our mRT-PCR assay is reliable for clinical diagnosis and rapid identification of CIVs.