Abstract：Abstract： The sequences of the HA and NA gene of the new pandemic A/H1N1 2009 influenza virus were obtained from GenBank. After multi-sequence alignment, two sets of specific PCR primers and TaqMan probes were designed. Two fluorescent probes were labeled with FAM and CY5 as reporter, respectively, and BHQ (black hole quencher) as quencher. A multiplex real-time RT-PCR assay was established to detect this new influenza A/H1N1 virus. The amplification curves of HA and NA gene of positive controls both exhibited standard S shape, suggesting a good amplification efficiency and linear relationship. Several other sub-types of influenza A virus samples including traditional H1N1, H3N2, H5N1, H6N2 and H9N2 were used as negative controls to validate this assay, for which no positive amplification signal was detected. The results indicated that our newly established assay had high specificity. In sensitivity test, 10 copies of plasmid DNA template can be detected, which nearly reach the detection limit. The whole multiplex real-time RT-PCR reaction could be finished in 90 min. 243 human clinical samples and 1351 swine clinical samples were examined with this assay. 7 human clinical samples were positive and all swine clinical samples were negative, which were consistent with the results from the test by using the commercial kits purchased from China academy of inspection and quarantine. Our study indicates that this newly established multiplex real-time RT-PCR assay is rapid, reliable and sensitive and it is suitable for application in new influenza A/H1N1 virus screening in laboratory.
