Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN, United States.
J Clin Virol. 2012 Aug;54(4):371-5. doi: 10.1016/j.jcv.2012.05.005. Epub 2012 Jun 6.
Human metapneumovirus (HMPV) is a paramyxovirus with multiple genetic lineages that is a leading cause of acute respiratory disease. Several RT-PCR assays have been described based on limited available sequence data.
To develop a broadly reactive real-time RT-PCR assay for HMPV that allows for a rapid, sensitive, and specific detection in a clinical or research setting.
Three published assays for HMPV were modified based on analysis of multiple HMPV sequences obtained from GenBank. Original and modified assays were tested against prototype HMPV strains from each genetic sublineage, multiple isolates of HMPV from different years, a collection of clinical specimens, and commercial validation panels.
A number of potential sequence mismatches with diverse HMPV strains were identified. Modifications were made to oligonucleotides to improve annealing efficiency. Primers and probes based on newer sequence data offered enhanced detection of all subgroups, especially for low titer specimens. The new primers and probe detected multiple clinical isolates of HMPV collected over a twenty-year period. The modified assay improved detection of HMPV in a panel of clinical specimens, and correctly identified HMPV samples in two commercial validation sets.
We report a modified real-time RT-PCR assay for HMPV that detects all genetic lineages with high sensitivity.
人偏肺病毒（HMPV）是一种副黏病毒，具有多个遗传谱系，是急性呼吸道疾病的主要原因。已经基于有限的可用序列数据描述了几种 RT-PCR 检测方法。
开发一种广泛反应性的 HMPV 实时 RT-PCR 检测方法，以便在临床或研究环境中进行快速、敏感和特异性检测。
根据从 GenBank 获得的多个 HMPV 序列分析，对三种已发表的 HMPV 检测方法进行了修改。原始和修改后的检测方法针对每个遗传亚谱系的原型 HMPV 株、来自不同年份的多种 HMPV 分离株、临床标本集和商业验证面板进行了测试。
鉴定出与多种 HMPV 菌株存在潜在序列不匹配的情况。对寡核苷酸进行了修改以提高退火效率。基于更新的序列数据的引物和探针提高了对所有亚群的检测能力，特别是对低滴度标本的检测能力。新的引物和探针检测了过去 20 年收集的多种临床分离株的 HMPV。改进后的检测方法提高了临床标本中 HMPV 的检测能力，并在两个商业验证组中正确识别了 HMPV 样本。
我们报告了一种改良的 HMPV 实时 RT-PCR 检测方法，该方法具有高灵敏度，可以检测所有遗传谱系。
