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.

Abstract

Two real time one-step RT-PCR assays were developed for simultaneous detection and typing of influenza A and B viruses and detection of Respiratory Syncytial Virus (RSV). As regard influenza, primers were designed to amplify specific sequences of gene M of A/H1N1, A/H3N2, A/H5N1, A/H7N7 and A/H9N2 viruses and of gene NP of type B viruses belonging both Yamagata and Victoria lineage. Specificity, analytical and clinical sensitivity, dynamic range, linearity of the new assays were evaluated. Dynamic ranges for Influenza A and B, and RSV were at least five logs and linearity was conserved. In order to evaluate the specificity, 80 nasopharyngeal swabs resulting Influenza and RSV negative by multiplex nested PCR and cell 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, 3 A/H1N2, 8 A/H3N2 Johannesburg/94-like, 10 A/H3N2 Panama/2007/99-like, 10 A/H3N2 Fuijian/411/02-like, 2 A/H5N1, 2 A/H7N7 and 2 A/H9N2, 15 B/Yamagata-like and 13 B/Victoria-like) collected between 1994 and 2004 or received by WHO Influenza Centre, London, were chosen as representative of the circulating strains and tested. All samples resulted positive although one B/Victoria sample was not clear typed. Thirty swabs nested RT-PCR positive for RSV collected during the four seasons, were also analysed by realtime PCR, resulting positive. To evaluate the performance of the new assay on fresh material, 250 specimens, collected during the 2004/05 seasons, were tested by nested-PCR, cell culture and real-time PCR. The new assays provide accurate and sensitive diagnosis of influenza and RSV infection and they represent a sensitive tool for virological surveillance and management of patient with ILI.