山羊痘病毒TaqMan-MGB荧光定量PCR检测方法的建立与应用

Development and application of TaqMan-MGB real-time quantitative PCR assay for detection of goat pox virus

摘要:

利用DNAStar 分析了GenBank中所有8株羊痘病毒全基因序列, 选取位于山羊痘病毒(AY077835)基因组gp064区域约64 bp的基因片段, 设计并合成了一对PCR引物和一条TaqMan-MGB探针, 建立了FQ-PCR和标准曲线,并利用该方法对山羊痘临床皮肤痘疹材料和人工感染动物材料进行GPV核酸检测。结果表明, 构建的FQ-PCR具有良好的敏感性、特异性、稳定性和临床应用性。该方法的建立为山羊痘临床快速高效地诊断和山羊痘病毒感染羊只的发生、发展及转归研究提供了一种有效的手段。

Abstract:

The complete gene sequences of eight capripoxvirus strains in GenBank were aligned and analyzed with DNAStar software. We selected a size of 64 bp gene fragment that was located in gp064 region of goat pox virus (GPV) genome, and designed a pair of primers and a TaqMan-MGB probe against the gene fragment with Primer Express 2.0 software. Then, the fluorescence quantitative PCR (FQ-PCR) assay was developed and the standard curve of different dilution series was described. We extracted the DNA samples from clinical skin pox, scab and GPV infected materials of artificial challenge animals. The FQ-PCR assay has been performed for all kinds of DNA samples. The results showed that the FQ-PCR assay was sensitive, specific, stable and could be used for clinical diagnosis. This method provided an important tool for rapid diagnosis of goat pox clinically, and for study GPV pathogenesis in the course of disease occurrence, development and convalescence.