Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV- 1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex I and the hemi-nested multiplex I detected I and 0. 1 TCID50 of RSV A, respectively, and 0.01 and 0.00 1 TCID50 of influenza virus AWN2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/9 1) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also-detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses. (c) 2005 Elsevier B.V. All rights reserved.