Performance testing of two new one-step real time PCR assays for detection of human influenza and avian influenza viruses isolated in humans and Respiratory Syncytial Virus

Introduction and Methods. Two real time one-step RT-PCRassays were developed for simultaneous detection and typingof influenza A and B viruses and detection of Respiratory Syncytial Virus (RSV). As regard influenza, primers were designedto amplify specific sequences of gene M of A/H1N1, A/H3N2,A/H5N1, A/H7N7 and A/H9N2 viruses and of gene NP of typeB viruses belonging both Yamagata and Victoria lineage. Specificity, analytical and clinical sensitivity, dynamic range, linearity of the new assays were evaluated.Results. Dynamic ranges for Influenza A and B, and RSV,were at least five logs and linearity was conserved. In orderto evaluate the specificity, 80 nasopharyngeal swabs resulting Influenza and RSV negative by multiplex nested PCR andcell culture, were tested and 79 resulted negative. The detection limits for influenza A and B, calculated by 95% probit,was 0,008 and 0,09 PFU, respectively, resulting more sensible than nested PCR. A total of 75 specimens (10 A/H1N1,