Duplex real-time RT-PCR assay for detection and subgroup-specific identification of human respiratory syncytial virus
Duplex real-time RT-PCR assay for detection and subgroup-specific identification of human respiratory syncytial virus
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DOI：
10.1016/j.jviromet.2019.113676
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发表时间：
2019-09-01
影响因子：
3.1
通讯作者：
Peret, Teresa C. T.
中科院分区：
文献类型：
--
作者：
Wang, Lijuan;Piedra, Pedro A.;Peret, Teresa C. T.
人呼吸道合胞病毒（HRSV）是全球幼儿急性呼吸道疾病的主要原因。在预期新型HRSV疫苗和免疫疗法获得许可的情况下，HRSV亚群A和B感染的可靠检测和鉴定对于准确估计疾病负担至关重要。为了确保持续的可靠性，分子检测必须与不断发展的病毒株保持同步。我们已经开发了一种HRSV亚组特异性实时RT-PCR（rRT-PCR）检测和亚组鉴定使用引物和亚组特异性探针靶向的核蛋白基因的保守区域结合在一个单一的双重反应，使用所有基因组序列数据，目前在GenBank中。通过与已建立的pan-HRSV rRT-PCR参考检测试剂直接比较，验证了该检测试剂盒的分析灵敏度、特异性、重现性和临床性能，并采集了不同地理位置的病毒分离株和呼吸道标本。该测定是灵敏的，可重复地检测靶RNA的少至5-10个拷贝/反应。该测定具有特异性，显示16种其他常见呼吸道病原体的组无扩增或通过计算机引物/探针分析预测。基于最新可用基因组序列数据的双重rRT-PCR检测方法可快速、灵敏和特异地检测HRSV并鉴定其亚组。
Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Reliable detection and identification of HRSV subgroup A and B infections are essential for accurate disease burden estimates in anticipation of licensure of novel HRSV vaccines and immunotherapies. To ensure continued reliability, molecular assays must remain current with evolving virus strains. We have developed a HRSV subgroup-specific real-time RT-PCR (rRT-PCR) assay for detection and subgroup identification using primers and subgroup-specific probes targeting a conserved region of the nucleoprotein gene combined in a single duplex reaction using all genome sequence data currently available in GenBank. The assay was validated for analytical sensitivity, specificity, reproducibility, and clinical performance with a geographically diverse collection of viral isolates and respiratory specimens in direct comparison with an established pan-HRSV rRT-PCR reference test. The assay was sensitive, reproducibly detecting as few as 5-10 copies/reaction of target RNA. The assay was specific, showing no amplification with a panel of 16 other common respiratory pathogens or predicted by in silico primer/probe analysis. The duplex rRT-PCR assay based on the most current available genome sequence data permits rapid, sensitive and specific detection and subgroup identification of HRSV.