OBJECTIVETo establish and evaluate a single-tube multiplex RT-real time PCR assay for detecting novel influenza A H1N1, influenza A and influenza B viruses (called "IV" for short) simultaneously.

METHODSA total of 213 clinical specimens of influenza-like patient's throat swab were collected during October 2010 and April 2011. 152 bp fragment in HA gene of novel influenza A H1N1 virus, 128 bp fragment in M gene of influenza A virus and 107 bp fragment in NP gene of influenza B virus were chosen as the target genes for multiplex RT-real time PCR, a specific primers and probes labeled with different fluoresceins were designed. The standard plasmid was constructed using in vitro transcription assay, and the standard curve was established. The reproducibility, specificity and sensitivity of the assay were evaluated. Furthermore, RNA extracted from 213 clinical specimens of throat swab was detected and verified by sequencing.

RESULTSThe corresponding standard curves of novel influenza A H1N1 virus, influenza A virus and influenza B virus were Y = - 3.46 lgX + 46.985, Y = - 3.49 lgX + 37.709, Y = - 3.51 lgX + 38.889, respectively; Y was cycle threshold (Ct), and lgX was logarithm value of virus replication number. The standard curve coefficient was 0.998. The detection limit of this assay was 10(2) copies/microl in one reaction. The specificity was strong. 39 (18.3%), 63 (29.6%) and 23 (10.8%) of 213 clinical specimens detected were positive for novel influenza A H1N1 virus RNA,influenza A virus RNA and influenza B virus RNA respectively. The positive samples were verified by sequencing.

CONCLUSIONThe single-tube multiplex RT-real time PCR assay developed in this study for detecting and identifying novel influenza A H1N1, influenza A and influenza B viruses simultaneously was rapid, specific and sensitive.