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猪种、牛种、羊种、绵羊种布鲁菌多重PCR检测方法的建立及应用
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引用本文:何亚鹏,许信刚,付明哲,张 琪.猪种、牛种、羊种、绵羊种布鲁菌多重PCR检测方法的建立及应用[J].西北农业学报,2017,26(8):1135~1140
DOI:10.7606/j.issn.1004-1389.2017.08.005
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作者单位
何亚鹏,许信刚,付明哲,张 琪 (西北农林科技大学 动物医学院陕西杨凌 712100) 
基金项目:陕西省农业科技创新与攻关(2016NY-092);陕西省重点产业创新链(2016KTZDNY02-06);西北农林科技大学试验示范站(基地)科技成果推广(TGZX2015-32)。
中文摘要:为建立在临床样本能够同时检测猪种、牛种、羊种、绵羊种布鲁菌的多重PCR方法,根据NCBI已收录的布鲁菌全基因,设计并合成4对特异性引物,通过优化多重PCR的反应条件,建立能够同时检测4种布鲁菌的多重PCR诊断方法。特异性试验结果表明,可以从4种布鲁菌以及 4 种细菌混合物中扩增出大小分别为276、494、733和976 bp的特异性条带,对照组的检测结果为阴性;敏感性试验结果表明,对4种病原菌基因组DNA的检出量为猪种32.2 pg、牛种21.3 pg、羊种27.5 pg、绵羊种43.2 pg;人工模拟感染样本检测结果表明,能从混合感染的病料中特异地检测出 4 种病原菌。应用该方法对 200 份临床奶山羊乳样进行检测,结果检出4份阳性。建立的多重PCR方法具有特异性强、敏感度高、稳定性好的特点,可以有效地检测 4 种布鲁菌的感染。
中文关键词:猪种布鲁菌  牛种布鲁菌  羊种布鲁菌  绵羊种布鲁菌  多重PCR
 
Foundation and Initial Application of Multiplex PCR Method for Detecting B.suis, B.abortus, B.melitensis and B.ovis Infection
Abstract:In order to develop multiplex PCR assay for clinical detection of B.suis, B.abortus, B.melitensis and B.ovis infection, four pairs of primers were designed and synthesized based on highly conserved regions of B.suis, B.abortus, B.melitensis and B.ovis genome sequence in Genbank. The multiplex PCR reaction condition was optimized and multiplex PCR method for detecting B.suis, B.abortus, B.melitensis and B.ovis infection was established. The specificity test showed that fragments of 276 bp, 494 bp, 733 bp and 976 bp were amplified from genomic DNA of B.suis, B.abortus, B.melitensis and B.ovis,respectively. No amplification was achieved from control groups of other bacteria. The sensitivity test showed that the multiplex PCR could detect genome DNA of 21.3 pg for B.abortus, 27.5 pg for B.melitensis, 43.2 pg for B.ovis and 32.2 pg for B.suis. The artificial simulation test showed that the multiplex PCR could detect four kinds of pathogens from co-infection disease material. The initial application test showed 200 clinical goat’s milk samples were subjected to PCR. Among 4 clinical samples were B.melitensis-positive. The result indicated the multiplex PCR method had advantages of specificity, sensitivity, repetition, and it could effectively detect the infection of B.suis, B.abortus, B.melitensis and B.ovis in clinical samples.
keywords:B.suis  B.abortus  B.melitensis  B.ovis  Multiplex PCR
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