弓形虫半巢式PCR检测方法的建立  

Establishment of the hemi-nested PCR for detecting Toxoplasma gondii

作　　者：孔得翔[1] 王素华 曲道峰[1] 蔡渭明 杜爱芳[1] 

机构地区：[1]浙江大学动物预防医学研究所,浙江杭州310029 [2]温州出入境检验检疫局,浙江温州325000 [3]浙江出入境检验检疫局,浙江杭州310004

出　　处：《中国兽医学报》2009年第1期52-54,62,共4页Chinese Journal of Veterinary Science

基　　金：浙江省科技厅重点项目(2006C22043);国家质检总局资助项目(2005J0013)

摘　　要：根据GenBank上公布的弓形虫RH株的P30基因(X14080)设计了3条特异性引物P1、P2和P3,建立了半巢式PCR检测体系,并将P1和P3的扩增产物克隆到pUCm-T载体上进行测序。结果表明,该体系能够扩增出约250 bp的片段,P1和P3能扩增出约500 bp的片段,克隆到pUCm-T载体上的504 bp片段与P30基因(X14080)的序列同源性为99.8%。该PCR检测体系特异性强,在健康猪血液、猪链球菌、多杀性巴氏杆菌、胸膜肺炎放线杆菌和猪圆环病毒DNA上未扩增出条带;敏感性高,最低检测为18 fg。该检测体系的成功构建为猪弓形虫的检测和流行病学调查提供了有力的技术支持。The primers were designed for the establishment of the hemi-nested PCR. The PCR product of primer P1 and P3 was ligated to the pUCm-T vector and sequenced. The product of the Hemi-nested PCR was about 250 bp and the product of primer P1 and P3 was about 500 bp. The 500 bp fragment ligated to the pUCm-T vector was 99. 8% identical to the published P30 gene（X14080）. The special test showed that no fragment was amplified while detecting nomal swine blood, Hemophiluspara suis, Pasteurellamultocida, Actinobacillus pneumoniae, PCV-2. Result of seneitivity showed the detection limit of this method was 18 fg. This specific and sensitive method is suitable for the detection of T. gondii and give proofs for the research of epidemiological analysis.

关 键 词：猪弓形虫病 P30基因 半巢式PCR 克隆 

分 类 号：S852.7[农业科学—基础兽医学] S855.99[农业科学—兽医学]