Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested‐PCR assay

The clinical presentation of infections caused by the heterogeneous group of the respiratory viruses can be very similar. Thus, the implementation of virological assays that rapidly identify the most important viruses involved is of great interest. A new multiplex reverse transcription nested‐polymerase chain reaction (RT‐PCR) assay that is able to detect and type different respiratory viruses simultaneously is described. Primer sets were targeted to conserved regions of nucleoprotein genes of the influenza viruses, fusion protein genes of respiratory syncytial viruses (RSV), and hexon protein genes of adenoviruses. Individual influenza A, B, and C viruses, RSV (A and B), and a generic detection of the 48 serotypes of adenoviruses were identified and differentiated by the size of the PCR products. An internal amplification control was included in the reaction mixture to exclude false‐negative results due to sample inhibitors and/or extraction failure. Detection levels of 0.1 and 0.01 TCID50 of influenza A and B viruses and 1–10 molecules of cloned amplified products of influenza C virus, RSV A and B, and adenovirus serotype 1 were achieved. The specificity was checked using specimens containing other respiratory viruses and no amplified products were detected in any case. A panel of 290 respiratory specimens from the 1999–2000 and 2000–2001 seasons was used to validate the assay. Accurately amplifying RNA from influenza and RSV prototype strains and DNA from all adenovirus serotypes demonstrates the use of this method for both laboratory routine diagnosis and surveillance of all these viruses. J. Med. Virol. 69:132–144, 2003. © 2003 Wiley‐Liss, Inc.