Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds
Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds
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DOI：
10.1128/jcm.39.12.4456-4461.2001
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发表时间：
2001-12-01
影响因子：
9.4
通讯作者：
Garin, D
中科院分区：
文献类型：
--
作者：
Garcia, S;Crance, JM;Garin, D
裂谷热病毒（RVFV）是白蛉病毒属（布尼亚病毒科）的一员，是一种具有三方基因组的包膜负链RNA病毒。直到2000年，裂谷热病毒的传播仅限于非洲大陆，但最近在阿拉伯半岛发生的致命疫情极大地说明了快速诊断方法、有效治疗和预防的必要性。采用TaqMan技术，针对非结构蛋白编码区，建立了实时检测逆转录(RT)-PCR定量小RNA片段的方法，并设计了引物和探针。在优化扩增反应和用含有目标基因的质粒体外转录的合成RNA建立校准曲线后，用感染Vero细胞的RVFV样本进行实时RT-PCR评估。结果表明，该方法对RVFV具有特异性，并成功应用于RVFV感染动物血清中RVFV基因组的检测以及各种药物（利巴韦林、α干扰素、6-氮脲和甘草酸）的抗病毒活性评估。总之，结果表明，传染性病毒滴度与实时RT-PCR检测的病毒基因组数量之间存在很强的相关性。这种新方法具有敏感性和省时性，且不需要对感染性物质进行操作，可用于快速诊断和筛选新的抗病毒化合物。
The Rift Valley fever virus (RVFV), a member of the genus Phlebovirus (family Bunyaviridae) is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity. Altogether, the results indicated a strong correlation between the infectious virus titer and the amount of viral genome assayed by real time RT-PCR. This novel method could be of great interest for the rapid diagnosis and screening of new antiviral compounds, as it is sensitive and time saving and does not require manipulation of infectious material.