副猪嗜血杆菌荧光定量PCR检测技术的建立与应用  

Development and Application of A Fluorescent Quantitative PCR Assay for the Detection of Haempohlius Parasuis

作　　者：于江[1,2] 吴家强[2] 张玉玉[2] 王兆[1] 李俊[2] 丁鹏[1,2] 李坤[1,2] 刘洋[1,2] 张金强[2,3] 徐绍建[2] 刘少宁[2,3] 周顺[1] 王文志[2] 王金宝[1,2,3] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]山东省农业科学院畜牧兽医研究所,山东济南250100 [3]山东农业大学动物科技学院,山东泰安271000

出　　处：《家畜生态学报》2010年第4期77-81,共5页Journal of Domestic Animal Ecology

基　　金：国家转基因生物新品种培育科技重大专项(2009ZX08009-143B);山东省自然科学基金重点项目(Z2007D06);山东省中青年科学家科研奖励基金(2009BSC02016);山东省留学回国人员科技活动择优资助项目(鲁人社函[2009]341号)

摘　　要：根据GenBank中的副猪嗜血杆菌(HPS)的infB基因的序列(DQ781808.1),利用分子生物学软件Premier 5.0设计一对特异性引物,建立基于SYBR Green I荧光定量PCR检测HPS的技术。试验采用煮沸法从HPS培养物中提取DNA,进行PCR扩增。将鉴定正确的目的基因片段克隆入pMD18-T载体中,转化大肠杆菌DH5α,经PCR及测序鉴定后得到阳性重组质粒,作为阳性标准品。结果表明,该方法对HPS具有良好的特异性,不与其它猪源细菌发生交叉反应,其敏感性比常规PCR高100倍,而且非常稳定,批间与批内重复试验变异系数均小于2.5%。临床应用表明本方法的建立实现了对HPS的快速诊断和定量的检测。A fluorescent quantitative PCR based on SYBR Green I was developed for the detection of Haempohlius parasuis. A pair of primers were designed specificly for infB gene of HPS according to the published nucleotide sequence （GenBank accession No. ：DQ781808.1）. The expected fragment was amplified using PCR from extracted DNA of HPS culture by boiling. The PCR product of target gene was cloned into pMD18-T vector and then transferred into DH5α. Positive recombinant plasmid was selected and used as a positive quantitative template to establish a standard curve. The results showed that the fluorescence quantitative PCR assay was able to detect HPS and had no cross-reaction with other bacteria from swine. The sensibility of this assay attained 10 copies of plasmid DNA, which was 100 times higher than routine PCR. The C. V. of the intra or inter was no more than 2.5%, indicating that the developed fluorescence quantitive PCR is a stable assay. Clinical detection showed development of the method achieved the rapid diagnosis and quantitative detection of HPS.

关 键 词：副猪嗜血杆菌 infB基因 SYBR Green I 荧光定量PCR 

分 类 号：S811.5[农业科学—畜牧学]