副猪嗜血杆菌毒力菌株和猪支气管败血波氏杆菌毒力菌株双重PCR检测方法的建立及应用  

Establishment and application of duplex PCR for detecting virulent strains of Haemophilus parasuis and Bordetella bronchiseptica

作　　者：覃娟娟 顾凡[1] 蔡雨涵 乔小改[1] 刘鹏娟[1] 李萍[1] 朱玲[1,2] 徐志文[1,2] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安625014 [2]动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医学报》2015年第8期1217-1222,共6页Chinese Journal of Veterinary Science

基　　金：教育部长江学者和创新团队发展计划资助项目(IRT 13083);教育部新世纪优秀人才支持计划资助项目(NCET 11-1059)

摘　　要：为建立一种能同时检测副猪嗜血杆菌(Hps)毒力菌株和猪支气管败血波氏杆菌(Bb)毒力菌株快速、准确的诊断方法,依据毒力血清型Hps OMP P2和Bb DNT基因序列分别设计合成引物,通过优化反应条件,成功建立了同时检测Hps毒力菌株和Bb毒力菌株的双重PCR方法。本试验建立的双重PCR法特异性强,重复性好,最低可检测核酸质量浓度分别达到1.79×10-3 g/L及8.3×10-3 g/L。使用该方法对来自四川地区部分猪场的36份猪呼吸综合征(PRDC)病料进行检测,结果检出率分别是33.33%和5.56%,与单一PCR结果一致,但高于细菌分离鉴定的准确率。试验结果表明该方法具有临床实用性,为四川地区Hps和Bb的诊断和预防奠定基础。To establish a rapid and accurate diagnostic method for simultaneous detection of Haemophilus parasuis（Hps）virulent strains and Bordetella bronchiseptica（Bb）virulent strains,the primers based on the virulence of serotype Hps OMP P2 and Bb DNT gene sequences were designed and synthesized.By optimizing the reaction conditions,the duplex PCR method for detecting Hps virulent strains and Bb virulent strain was established successfully.The duplex PCR method was characterized by specificity and repeatability,the minimum detectable nucleic acid concentrations reached 1.79×10-3 g/L and 8.3×10-3 g/L,respectively.The duplex PCR method was applied to the detection of 36 PRDC disease samples from Sichuan province.The positive rates of Hps and Bb were 33.33%and 5.56%,consistent with those of a single PCR,but higher than the bacterial isolation.The method established by this experiment was very useful and can provide a basis for Hps and Bb diagnosis and prevention in the Sichuan region.

关 键 词：副猪嗜血杆菌 猪支气管败血波氏杆菌 毒力菌株 双重PCR 

分 类 号：S852.61[农业科学—基础兽医学]