Lineage-specific detection of influenza B virus using real-time polymerase chain reaction with melting curve analysis.

Abstract

Influenza B viruses comprise two lineages, Victoria (B/Vic) and Yamagata (B/Yam), which co-circulate globally. The surveillance data on influenza B virus lineages in many countries often underestimate the true prevalence due to the lack of a rapid, accurate, and cost-effective method for virus detection. We have developed a real-time PCR with melting curve analysis for lineage-specific differential detection of influenza B virus. By amplifying a region of the hemagglutinin gene using real-time PCR with SYBR Green I dye, B/Vic and B/Yam could be differentiated based on their melting temperature peaks. This method was efficient (B/Vic = 93.2%; B/Yam 97.7%), sensitive (B/Vic, 94.6%; B/Yam, 96.3%), and specific (B/Vic, 97.7%; B/Yam, 97.1%). The lower detection limit was 10(2) copies per microliter. The assay was evaluated using 756 respiratory specimens that were positive for influenza B virus, obtained between 2010 and 2015. The incidence of influenza B virus was approximately 18.9% of all influenza cases, and the percentage was highest among children aged 6-17years (7.57%). The overall percentage of mismatched influenza B vaccine was 21.1%. Our findings suggest that real-time PCR with melting curve analysis can provide a rapid, simple, and sensitive lineage-specific influenza B virus screening method to facilitate influenza surveillance.