Tewawong Nipaporn, Chansaenroj Jira, Klinfueng Sirapa, Vichiwattana Preeyaporn, Korkong Sumeth, Thongmee Thanunrat, Theamboonlers Apiradee, Payungporn Sunchai, Vongpunsawad Sompong, Poovorawan Yong
Center of Excellence in Clinical Virology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand.
Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Arch Virol. 2016 Jun;161(6):1425-35. doi: 10.1007/s00705-016-2802-2. Epub 2016 Feb 29.
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 6-17 years (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.
乙型流感病毒包括两个谱系，即维多利亚系（B/Vic）和山形系（B/Yam），它们在全球共同流行。由于缺乏一种快速、准确且经济高效的病毒检测方法，许多国家关于乙型流感病毒谱系的监测数据往往低估了其实际流行率。我们开发了一种带有熔解曲线分析的实时荧光定量PCR方法，用于乙型流感病毒谱系特异性差异检测。通过使用SYBR Green I染料的实时荧光定量PCR扩增血凝素基因区域，可根据B/Vic和B/Yam的熔解温度峰进行区分。该方法效率高（B/Vic = 93.2%；B/Yam = 97.7%）、灵敏度高（B/Vic为94.6%；B/Yam为96.3%）且特异性强（B/Vic为97.7%；B/Yam为97.1%）。最低检测限为每微升10²个拷贝。使用2010年至2015年间获得的756份乙型流感病毒阳性呼吸道标本对该检测方法进行了评估。乙型流感病毒的发病率约占所有流感病例的18.9%，在6至17岁儿童中该比例最高（7.57%）。乙型流感疫苗不匹配的总体比例为21.1%。我们的研究结果表明，带有熔解曲线分析的实时荧光定量PCR可为乙型流感病毒谱系特异性筛查提供一种快速、简单且灵敏的方法，以促进流感监测。
