猪附红细胞体实时荧光定量PCR检测方法的建立  

Establishment of a real-time fluorescent quantitative PCR assay for the detection of Eperythrozoon suis

作　　者：朱绍辉[1] 吴延功[2] 单虎[1] 王志亮[2] 姜同泉[1] 孙承英[2] 赵永刚[2] 

机构地区：[1]莱阳农学院动物科技学院,山东青岛266109 [2]中国动物卫生与流行病学中心,山东青岛266032

出　　处：《中国兽医科学》2007年第2期108-112,共5页Chinese Veterinary Science

基　　金：国家"十五"科技攻关计划项目(2004BA514A)

摘　　要：参照猪附红细胞体16 S rRNA基因序列设计了1对引物,分别以标准株和分离株的基因组DNA为模板,采用SYBR GreenⅠ实时荧光定量PCR方法检测猪附红细胞体,确定特异性产物的Tm值,同时做普通PCR。试验结果表明,特异性产物的Tm值为88.5℃,最低能检测到含0.036 fg/μL阳性质粒的标准品。以阳性质粒标准品10倍倍比稀释的模板进行实时荧光定量PCR,通过对反应体系和反应条件进行筛选和优化,建立了检测猪附红细胞体的标准曲线。建立的SYBR GreenⅠ实时荧光定量PCR检测方法显示了较好的特异性、敏感性和广谱性,为猪附红细胞体病的临床检测和流行情况调查提供了新的技术手段。A pair of primers were designed based on the 16 S rRNA sequence of Eperythrozoon suis, and a real-time fluorescent quantitative PCR（real-time FQ-PCR） assay was established for the detection of E. suis by using DNA extracted from positive strain and the ferret isolated as templates. Tm value of the specific production was confirmed to be 88.5 ℃. An ordinary PCR was also performed. This technique was highly sensitive with a detection limit of 0. 036 fg/μL of positive recombinant plasmid. After the reaction conditions and reaction systems of the real-time FQ-PCR were optimized, a standard curve was obtained with the positive recombinant plasmid diluted by 10 times as template. The developed real-time FQ-PCR using SYBR Green Ⅰ was specific,highly sensitive and had broad applications, and could be further used in clinic detection and epidemic investigation of E. suis.

关 键 词：猪附红细胞体 实时荧光定量PCR 荧光染料 

分 类 号：S852.64[农业科学—基础兽医学] Q503[农业科学—兽医学]