Advanced Yellow Fever Virus Genome Detection in Point-of-Care Facilities and Reference Laboratories
Advanced Yellow Fever Virus Genome Detection in Point-of-Care Facilities and Reference Laboratories
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DOI：
10.1128/jcm.01799-12
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发表时间：
2012-12-01
影响因子：
9.4
通讯作者：
Niedrig, Matthias
中科院分区：
文献类型：
--
作者：
Domingo, Cristina;Patel, Pranav;Niedrig, Matthias
已报道的黄热病病毒基因组检测方法受到灵敏度、特异性、毒株检测谱以及对流行地区基础设施简单的实验室的适用性方面的限制。我们描述了两种不同方法的开发，这些方法可对黄热病基因组进行灵敏和特异的检测：实时逆转录定量 PCR (RT-qPCR) 和使用相同引物探针组但基于解旋酶依赖性扩增技术 (RT-tHDA) 的等温方案。两种测定均使用黄热病细胞培养上清液以及加标样品和临床样品进行评估。我们证明了通过两种检测方法可以可靠地检测不同毒株的黄热病病毒，并提高灵敏度和特异性。 RT-qPCR 测定是具有实时 PCR 功能的参考或诊断实验室的强大工具，而等温 RT-tHDA 测定是现场或护理点实验室黄热病分子诊断早期扩增技术的有用替代方案。
Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.