An Inexpensive and Accurate Reverse Transcription-PCR–Melting Temperature Analysis Assay for Real-Time Influenza Virus B Lineage Discrimination

In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-T(m)), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (T(m)) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene. A total of 410 samples collected during the 2004, 2008, and 2010–2017 influenza seasons in Brazil were tested, and the lineages were correctly characterized using their melting profiles. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test; P < 0.0001, confidence interval = 95%), and the T(m) is not affected by viral load (Spearman correlation test; r = 0.287, P = 2.245 × 10(−9)). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.