Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus
Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus
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DOI：
10.1016/j.jviromet.2007.12.003
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发表时间：
2008-03-01
影响因子：
3.1
通讯作者：
Li, Jinming
中科院分区：
文献类型：
--
作者：
Ba, Jingyue;Li, Lin;Li, Jinming
本研究建立了一种基于taqman的快速、特异的一步实时定量反转录PCR （qRT-PCR）检测小反刍兽疫毒（PPRV）的方法。根据核衣壳蛋白基因序列设计引物和探针。实时qRT-PCR检测能够检测到来自非常不同地理区域（非洲、中东和亚洲）的PPRV分离株。通过纳入牛瘟病毒和其他麻疹病毒rna来评估该方法的特异性，但这些病毒在该方法中均未检测出阳性。实时qRT-PCR分析的灵敏度是通过构建内部PPRV cRNA来生成标准曲线来实现的。该方法的检出限为每个反应混合物8.1个RNA拷贝。该方法具有良好的组内和组间重复性。采用常规RT-PCR和qRT-PCR相结合的方法，对30份现场样品进行PPRV检测。实时qRT-PCR的检出率由46.7%提高到73.3%。本报告中描述的实时qRT-PCR检测可以在现场病例的组织样本中快速、特异和敏感地进行PPRV实验室检测。(C) 2007 Elsevier B.V.版权所有
In this study, a rapid and specific TaqMan-based, one-step real-time quantitative reverse transcription PCR (qRT-PCR) has been described for the detection of peste des petits ruminants virus (PPRV). Primers and probe were designed based on the nucleocapsid protein gene sequence. The real-time qRT-PCR assay was able to detect PPRV isolates from very distinct geographical areas (Africa, Middle East and Asia). The specificity of the assay was assessed by including rinderpest virus and other morbillivirus RNAs but none of these tested positive in the assay. The analytical sensitivity of the real-time qRT-PCR assay was achieved through the construction of an in-house PPRV cRNA for the generation of a standard curve. The detection limit of the assay was found to be 8.1 RNA copies per reaction mixture. The assay had excellent intra- and inter-assay reproducibility. In total 30 field samples were screened for the presence of PPRV by conventional RT-PCR in parallel with qRT-PCR. The detection rate increased from 46.7% to 73.3% by use of the real-time qRT-PCR. The real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PPRV in tissue samples from field cases. (C) 2007 Elsevier B.V. All rights reserved.