山羊痘病毒SYBR Green Ⅰ实时定量PCR检测方法的建立  

Development of SYBR Green Ⅰ Real-time PCR for Goat Pox Virus

作　　者：姚俊[1] 杨永军[2] 彭海生 陈官国 段国军[2] 鲁富有 李华春[1] 

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,云南昆明650224 [2]云南省丽江市华坪县畜牧兽医站,云南华坪674800 [3]云南省普洱市动物疫病预防控制中心,云南普洱665000

出　　处：《动物医学进展》2012年第7期32-39,共8页Progress In Veterinary Medicine

基　　金：云南省科技厅农业科技创新工程项目(2008LA019)

摘　　要：为了建立一种灵敏、特异、快速的山羊痘病毒实时定量检测方法,根据山羊痘病毒(AY077835)gp006基因序列,应用Beacon Designer 7.7软件设计合成一对PCR引物。将被检测的目的 DNA片段克隆PEASY-T1质粒,制备重组质粒标准品,以期建立以质粒拷贝数为单位的标准曲线。通过反应条件的优化及引物浓度的筛选,建立了山羊痘病毒SYBR GreenⅠ实时定量PCR(real-time PCR)检测方法,最佳引物浓度为400nmol/L。组内重复试验和组间重复试验变异系数均低于2%,最低检测限为1×101 copies/μL,上机检测时间在1h以内。用该方法对2011年流行于云南华坪、景谷及2003年流行于云南宣威、寻甸和昆明的山羊痘临床组织样品进行定量检测,结果送检组织样品抽提DNA中病毒含量分别为1.03×105、5.30×103、1.51×104、4.74×103、7.20×104 copies/μL。结果表明,所建立的real-time PCR具有灵敏、特异、快速的特点,适合于山羊痘临床组织样品的定量检测。In order to develop a fluorescence quantitative detection method, which is sensitive, specific, fast, safe and low-cost, for goat pox virus（GTPV）,one pair of specific primers for SYBR Green I real-time PCR were designed according to gp006 gene sequence of goat pox virus genome（AY386264） available in NCBI GenBank by Beacon Designer software （version 7.7）. In order to make a standard curve for quanti- tative detection of goat pox virus,the DNA fragment amplified by above primers was cloned into PEASY- TI plasmid and the recombinant plasmid was identified, proliferated, purified and counted. Through opti- mizing the reaction conditions and primer concentrations, SYBR Green I real-time PCR detection method for goat pox virus was established and the optimal concentration for primers were 400 nmol/L. Coefficientof variation of intra- and inter-assay were both less than 2%. The minimum detection limit was 1 × 10^1 copies/μL and the detection time was less than one hour. This developed method was used to quantitative- ly detect clinical samples of GTPV that prevailed in Huapin, Jinggu of Yunnan province in 2011 and in Xu- anwei, Xundian, Kunming of Yunnan province in 2003. The virus numbers in extracted DNA from the specimen were 1.03 ×10^5 copies/μL, 5.30 × 10^3 copies/μL, 1.51 × 10^4 copies/μL, 4. 74 × 10^3 copies/μL, 7.20×10^4 copies/μL,respectively. Those results showed that the established SYBR Green I real-time PCR assay was sensitive,specific, fast, safe and was suited for quantitative detection of goat pox virus in clinical samnles.

关 键 词：山羊痘病毒 SYBRGreenⅠ 实时定量PCR 

分 类 号：S852.654[农业科学—基础兽医学] Q789[农业科学—兽医学]