OBJECTIVETo develop a novel sensitive duplex real-time PCR assay for accurately identifying B/Yamagata and B/Victoria lineages of influenza virus type B.

METHODS50 HA (hemagglutinin) gene sequences coding for B/Yamagata and B/Victoria lineage, respectively, were randomly downloaded for GenBank and analyzed by software MEGA. Primers and probes specific for HA gene of B/Yamagata and B/Victoria lineages were designed by Primer Primer and then applied in the duplex real-time RT-PCR method that was followed developed. Influenza virus B type and A type isolated in our laboratory and typing-confirmed by HAI method were used as reference strains to determine the specificity of this assay and the sensitivity of the duplex amplification was evaluated by viral load testing in terms of in vitro transcribed RNA copy number.

RESULTSIn 2006-2010, 793 influenza virus type B strains were isolated from 17 765 throat swab samples, among which 152 strains were differentiated as By lineage and 641 as Bv lineage by this assay. These results was agreement with that determined by HAI assay. This developed assay allows to accurately identify approximately 10(2) copies/microl for Bv and By lineage virus with intra- and inter-coefficient of variation (CV) < 3.5% and nearly 100% specificity.

CONCLUSIONSThis method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination of influenza virus, which could be applied in influenza surveillance laboratories for rapid molecular diagnosis.