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

J Clin Microbiol. 2019 Nov 22;57(12):e00602-19. doi: 10.1128/JCM.00602-19. Print 2019 Dec.

Abstract

In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm ), 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 (Tm ) 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 Tm 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.

Keywords: RT-PCR; Victoria lineage; Yamagata lineage; influenza B; melting curve; melting temperature.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brazil
  • Costs and Cost Analysis
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics
  • Humans
  • Influenza B virus / classification*
  • Influenza B virus / genetics
  • Influenza B virus / isolation & purification*
  • Influenza, Human / diagnosis*
  • Influenza, Human / virology
  • Nucleic Acid Denaturation*
  • Real-Time Polymerase Chain Reaction / economics
  • Real-Time Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / economics
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Time Factors

Substances

  • Hemagglutinin Glycoproteins, Influenza Virus