Highly sensitive and broadly reactive quantitative reverse transcription-PCR assay for high-throughput detection of rift valley fever virus
Highly sensitive and broadly reactive quantitative reverse transcription-PCR assay for high-throughput detection of rift valley fever virus
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DOI：
10.1128/jcm.00936-07
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发表时间：
2007-11-01
影响因子：
9.4
通讯作者：
Nichol, Stuart T.
中科院分区：
文献类型：
--
作者：
Bird, Brian H.;Bawiec, Darcy A.;Nichol, Stuart T.
裂谷热病毒是一种蚊媒病毒，与整个非洲和阿拉伯半岛的大规模兽疫/流行病有关。病毒感染可导致经济上灾难性的“流产风暴”和牲畜新生儿的高死亡率。人类感染会导致类似流感的疾病，1%至2%的患者会出现严重的并发症，包括脑炎或出血热，死亡率很高。目前迫切需要一种能够检测裂谷热病毒自然遗传谱的高度敏感和特异的分子诊断检测方法。我们在此报告建立了一种泛裂谷热病毒定量实时逆转录-PCR检测方法，该方法具有高分析灵敏度（相当于5个体外转录RNA拷贝/反应或相当于0.1 PFU感染性病毒/反应）和高效率（标准曲线斜率= -3.66）。基于56年（1944年至2000年）收集的40种生态和生物多样性病毒分离株的完整基因组序列的比对，已知本试验中靶向的引物和探针退火位点位于高度保守的基因组区域。通过检测2000年沙特阿拉伯暴发期间获得的已知裂谷热病例材料，说明了这种检测方法相对于血清学检测方法的性能。此外，对那次疫情期间从人类患者（25例非致命性，8例致命性）中收集的急性期血液样本的分析表明，患者在医院就诊时的病毒血症可能是确定患者结局的有用预后工具。
Rift Valley fever (RVF) virus is a mosquito-borne virus associated with large-scale epizootics/epidemics throughout Africa and the Arabian peninsula. Virus infection can result in economically disastrous "abortion storms" and high newborn mortality in livestock. Human infections result in a flu-like illness, with 1 to 2% of patients developing severe complications, including encephalitis or hemorrhagic fever with high fatality rates. There is a critical need for a highly sensitive and specific molecular diagnostic assay capable of detecting the natural genetic spectrum of RVF viruses. We report here the establishment of a pan-RVF virus quantitative real-time reverse transcription-PCR assay with high analytical sensitivity (similar to 5 RNA copies of in vitro-transcribed RNA/reaction or similar to 0.1 PFU of infectious virus/reaction) and efficiency (standard curve slope = -3.66). Based on the alignments of the complete genome sequences of 40 ecologically and biologically diverse virus isolates collected over 56 years (1944 to 2000), the primer and probe annealing sites targeted in this assay are known to be located in highly conserved genomic regions. The performance of this assay relative to serologic assays is illustrated by testing of known RVF case materials obtained during the Saudi Arabia outbreak in 2000. Furthermore, analysis of acute-phase blood samples collected from human patients (25 nonfatal, 8 fatal) during that outbreak revealed that patient viremia at time of presentation at hospital may be a useful prognostic tool in determining patient outcome.