副猪嗜血杆菌ERIC-PCR分型方法的建立及其应用  

Establishment and application of ERIC-PCR method for genotyping of in Haemophilus parasuis

作　　者：郑念广[1] 谭实勇[2] 陈瑞爱[1,3] 何玲[3] 夏芳[3] 罗满林[1,3] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]广东华农温氏畜牧股份有限公司,广东云浮527400 [3]广东大华农动物保健品股份有限公司,广东云浮527400

出　　处：《中国兽医科学》2011年第4期347-350,共4页Chinese Veterinary Science

基　　金：广东温氏食品集团有限公司2008年度第八批科研项目(C080921)

摘　　要：利用ERIC序列设计1对引物,分别对PCR反应的模板量、引物浓度、DNA聚合酶用量、dNTPs浓度、退火温度等影响ERIC-PCR扩增的因素进行了探索和优化,以选择最佳的PCR反应体系及反应条件。结果显示,确定的最适引物浓度为0.166～0.333μmol/L,DNA聚合酶的最适浓度为1.0U/μL,模板最适用量为8ng/μL,dNTPs为200μmol/L。利用所建立的副猪嗜血杆菌ERIC-PCR分子生物学分型技术,对临床分离菌株的指纹图谱进行了分析,13个分离菌株的ERIC-PCR指纹图谱与15个标准血清型指纹图谱相比较,可分辨出6种血清型,并与琼脂扩散试验分型方法的结果一致。证实已建立的副猪嗜血杆菌ERIC-PCR分型方法重复性好、可比性高。A pair of primers were designed according to the ERIC sequence.The reaction system including the concentrations of templates,primers,DNA polymerase,dNTPs and the annealing temperature were explored and optimized for development of an ERIC-PCR method.Fingerprints of clinical isolates were analyzed by the established ERIC-PCR method for genotyping of Haemophilus parasuis.In result,the optimal concentration of primers was from 0.166 μmol/L to 0.333 μmol/L,the concentration of DNA polymerase was 1.0 U/μL.The concentration of template DNA was 8 ng/μL,and dNTPs was 200 μmol/L in content.Using the method,ERIC-PCR fingerprinting of 13 isolates compared with the 15 standard serum-based fingerprint could be distinguished six kinds of serotypes.These results were consistent with that of serological agar diffusion testing method.The results showed that the established ERIC-PCR technique for genotyping of H.parasuis had good reproducibility and high comparability.

关 键 词：副猪嗜血杆菌 肠杆菌科基因间重复序列为引物的聚合酶链反应 分型 

分 类 号：S852.612[农业科学—基础兽医学]