禽网状内皮组织增殖病病毒SYBR GreenⅠ实时荧光PCR检测方法的建立

Development of SYBR GreenⅠReal-time PCR for the Detection of Avian Reticuloendotheliosis Virus

为了探究禽网状内皮组织增殖病的快速检测方法,根据禽网状内皮组织增殖病病毒(REV)LTR和gag基因的保守区域各设计并合成一对引物,建立检测插入感染与全病毒感染的SYBR Green I模式的实时荧光PCR方法(Real-time PCR)。以pMD-LTR,pMD-gag重组质粒为标准品建立标准曲线,并对该方法的特异性、敏感性、重复性进行评价。结果表明:该方法线性关系良好;敏感性能检测到10拷贝标准品;能特异性检测REV样品,不与其它禽相关病原发生交叉反应;板内、板间重复试验的变异系数均低于3%。应用该方法对人工感染REV的鸡的心、肝、脾、肺、肾、盲肠扁桃体、腺胃和法氏囊进行初步检测,结果显示法式囊中的含量最高。

In order to develop rapid detection method for Avian Reticuloendotheliosis Virus（REV）,According to the REVgaggene sequence deposited in GenBank,apair of specific primers were designed which target to the conserved region of LTR gene and gaggene,a SYBR Green I-based real-time PCR method was established.The standard curve was constructed by using the pMD-18T-LTR and pMD-18T-gag plasmid.The results showed that high specificity as only the REV positive samples were amplified,and no cross-reaction was found with other avian pathogens.The coefficients of variation of intra-and inter-assay reproducibility were both less than 3%.The virus loads in these organs showed no evidently difference except bursa.