山羊淋巴结炎病原菌的分离鉴定及多重PCR检测方法的建立

发布时间：2018-09-04 05:56

【摘要】：旨在对重庆地区山羊体表型淋巴结炎病原菌进行分离鉴定,并建立相应检测方法。无菌采集山羊患病部位脓液,进行病原菌分离鉴定,依据菌株16SRNA基因序列构建系统进化发育树,同时设计特异性引物,建立PCR检测方法。结果表明,从病料中主要分离到3种病原菌,分别为伪结核棒状杆菌、化脓隐秘杆菌和金黄色葡萄球菌;依据伪结核棒状杆菌磷脂酶D、化脓隐秘杆菌16SRNA和金黄色葡萄球菌nuc保守核苷酸序列设计引物,建立的多重PCR检测方法,可以从3种菌株混合基因组中扩增出大小分别为507、1 378和278bp的对应菌株特异性序列,该方法最低检出浓度为103cfu·mL-1;对采集的80份临床样品进行多重PCR检测,发现伪结核棒状杆菌与金黄色葡萄球菌混合感染检出率为12.5%,伪结核棒状杆菌与化脓隐秘杆菌混合感染检出率为3.75%,三种菌混合感染检出率为7.5%。成功分离到山羊淋巴结炎的3种主要病原菌,并建立多重PCR检测方法,为该病的防控及治病机制研究奠定了基础。
[Abstract]:The aim of this study was to isolate and identify the pathogenic bacteria of goat body surface lymphadenitis in Chongqing area and to establish a corresponding detection method. The pathogenic bacteria were isolated and identified from the abscess collected from the diseased part of goat. The phylogenetic tree was constructed according to the 16SRNA gene sequence of the strain. At the same time, specific primers were designed to establish the detection method of PCR. The results showed that three kinds of pathogenic bacteria were mainly isolated from the diseased materials, which were Corynebacterium pseudotuberculosis, Stereobacterium pyogenes and Staphylococcus aureus. The primers were designed according to the phospholipase D of Corynebacterium pseudotuberculosis, the conserved nucleotide sequence of 16SRNA and nuc of Staphylococcus aureus, and the multiplex PCR detection method was established. The specific sequences of the corresponding strains were amplified from the mixed genomes of the three strains, and the corresponding specific sequences were obtained, respectively. The lowest detection concentration of this method was 103cfu mL-1; for multiple PCR detection of 80 clinical samples. It was found that the detection rate of mixed infection of Corynebacterium pseudotuberculosis and Staphylococcus aureus was 12.5, the detection rate of mixed infection of Corynebacterium pseudotuberculosis and Mycobacterium pyogenes was 3.75, the detection rate of mixed infection of three kinds of bacteria was 7.5%. Three major pathogens of goat lymphadenitis were successfully isolated and a multiplex PCR detection method was established, which laid a foundation for the study of the prevention and control of the disease and the mechanism of its treatment.
【作者单位】：重庆市畜牧科学院;重庆市畜牧科学院武陵分院;
【基金】：重庆市基础与前沿研究计划项目(cstc2015jcyjA80035) 国家自然科学基金(31402149)
【分类号】：S858.27

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