Robert Koch Institute, Center for Biological Security 1 (ZBS1), Nordufer 20, Berlin, 13353, Germany.
Virol J. 2013 Feb 14;10:58. doi: 10.1186/1743-422X-10-58.
The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.
A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively.
Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10-100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays.
The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.
黄病毒属包括几种能引起人类严重疾病的病原体。西尼罗河病毒在欧洲再次出现，以及某些黄病毒（如登革热、黄热病和日本脑炎病毒）的持续传播，对全球公共卫生构成了威胁。因此，需要一种快速、准确的分子方法来诊断和监测黄病毒。
本研究开发并优化了一种使用针对黄病毒 NS5 基因的锁定核酸探针的 Pan-Flavi 定量 RT-PCR 检测方法，以同时检测广泛的黄病毒。使用不同的黄病毒和非黄病毒 RNA 测试了 Pan-Flavi 检测方法的特异性和敏感性。此外，还直接比较了该检测方法与黄病毒种特异性检测方法检测黄病毒的敏感性。
设计了两个简并引物和一个锁定核酸探针，以扩增大多数黄病毒。为了提高 Pan-Flavi 检测方法对黄热病病毒和登革热病毒 4 的特异性和荧光信号，还包括了其他的引物和探针。验证了该检测方法的广谱特异性，可检测到 30 种不同的黄病毒 RNA。使用黄热病病毒疫苗株、登革热病毒 1 和蜱传脑炎病毒的标准质粒和 RNA 稀释液成功地进行了该检测方法的测试，灵敏度限值为 10-100 基因组拷贝/反应。与黄病毒种特异性检测方法相比，Pan-Flavi 检测方法在检测不同黄病毒时也取得了较好的结果。
该检测方法快速、广谱、特异性高、灵敏度高，是快速检测牲畜样本中黄病毒、流行病学研究或作为临床诊断中有用的黄病毒种特异性检测方法的补充的有价值的工具。
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