Multiplex RT-PCR for typing and subtyping of human influenza viruses

Objective To develop a multiplex reverse transcription-polymerase chain reaction (RT-PCR) to simultaneously type human influenza A virus and influenza B virus,and to subtype H1,H3,H5 and novel H1(swine H1) of human influenza virus type A.Method Primers for typing influenza viruses were targeted to conserved regions and a multiplex RT-PCR for typing and subtyping human influenza viruses was established.Results The type-specific primers were designed to amplify MA fragments of 214 bp for influenza A virus,and 296 bp for influenza B virus.The subtype-specific primers were designed to amplify HA fragments of 362 bp for H1,112 bp for H3,188 bp for H5 and 494 bp for novel H1,respectively.The sensitivity of the multiplex RT-PCR was evaluated by testing the novel H1N1 strain (A/Beijing/501/2009) and the synthetic full-length H1 gene of novel H1N1 virus(A/California/04/2009).The testing limit was 0.5 TCID50/reaction for the novel H1N1 virus.Clinical throat swab specimens collected from 39 suspected cases of novel A H1N1 patients were tested to validate the multiplex assay.Four of the specimens (10.2%) were confirmed as novel A (H1N1) positive,two were identified as seasonal H1N1 (5.1%) and 12 cases as H3N2 (30.7%),which were identical to real-time PCR assay recommended by WHO.Conclusion The developed multiplex RT-PCR assay is rapid,sensitive and specific for typing and subtyping human influenza viruses.