副猪嗜血杆菌PCR检测方法的建立及应用  

Establishment and Preliminary Application of PCR for Detecting Haemophilus parasuis

作　　者：梁乔[1,2] 杨本勇 石霖[1,2] 李井春 张健[1,2] 赵凤菊 Liang Qiao;Yang Benyong;Shi Lin;Li Jingchun;Zhang Jian;Zhao Fengju(Liaoning Center for Animal Disease Control and Prevention,Liaoning Shenyang 110164;Animal Medicine Research Institute of Liaoning Province,Liaoning Shenyang 110164)

机构地区：[1]辽宁省动物疫病预防控制中心,辽宁沈阳110164 [2]辽宁省动物医学研究院,辽宁沈阳110164

出　　处：《现代畜牧兽医》2018年第9期10-13,共4页Modern Journal of Animal Husbandry and Veterinary Medicine

摘　　要：为建立一种快速鉴别诊断副猪嗜血杆菌的方法,本试验针对副猪嗜血杆菌infB基因序列,利用Premier5.0软件设计并合成了1对特异性引物,建立了针对副猪嗜血杆菌的PCR鉴别诊断方法,优化了退火温度,检测了该方法的敏感性和特异性,结果表明,本试验建立的PCR检测方法的最佳退火温度为49℃。敏感性试验表明,该PCR检测方法最低能检测到的DNA浓度为18.1×10-6μg/mL。通过特异性试验表明,本方法仅可由副猪嗜血杆菌中扩增出特异性条带,在金黄色葡萄球菌、沙门氏菌等病原菌中扩增不出相应的条带,表明该方法具有较高的特异性。同时,通过对本方法检测出的阳性样品infB序列分析发现,与GenBenk中公布的副猪嗜血杆菌infB序列同源性均在98.2%~99%。表明建立的方法灵敏度高、特异性强,可以应用于副猪嗜血杆菌的实验室诊断。A PCR method for rapid differential diagnosis of Haemophilus parasuis was estab - lished. To provide effective technical support for the rapid diagnosis and epidemiological inves- tigation of Haemophilus parahaemophilus in pigs, and to overcome the shortcoming of poor speci - ficity of traditional identification methods. The sequence of infB gene of Haemophilus parahae- mophilus was analyzed. A pair of specific primers were designed and synthesized by Premier5.0 software. A method for differential diagnosis of Haemophilus parais by PCR was established. The optimum annealing temperature was determined to be 49 ℃ by multiple experiments. The lowest con - centration of DNA was18.1×10-6 g/mL by sensitivity test. At the same time, the infB sequence analysis of positive samples detected by this method showed that the homology of infB sequence with other Haemophilus parahaemophilus in GenBenk was above 98.2%～99%, which indicated that the sensitivity of the method was high. It can be used in laboratory diagnosis of Haemophilus parasu- is.

关 键 词：副猪嗜血杆菌 聚合酶链反应 建立 

分 类 号：S858.28[农业科学—临床兽医学]