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

Isolation and Identification of Pathogenic Bacteria of Lymphadenitis in Goat and Establishment of Multiplex PCR Detection Method

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。

The purpose of this paper was to isolate and identify the pathogenic bacteria of lymphadenitis in goat of Chongqing area and establish the test method.Sterile separation operation was carried out to isolate pathogenic bacteria from fester of the goat.The phylogenetic tree was founded according to the gene sequence of 16 SRNA.And the specific primers were designed to establish PCR test method.The result showed that Corynebacterium pseudotuberculosis,Arcanobacterium pyogenes and Staphylococcus aureus were isolated from the samples,and three pairs of specific oligonucleotide primers were respectively designed according to the gene sequence of Phospholipases D,16 SRNA and nucfor multiple PCR detection.The multiple PCR detection showed that a fragment of 507,1 378 and 278bp amplified from the mixture genome.The sensitivity test showed that the multiplex PCR could detect minimum detectable concentration of 103cfu·mL-1.The detection of 80 clinical samples indicated that,the detection rates of mixed infections ofCorynebacterium pseudotuberculosis and Staphylococcus aureus,Corynebacterium pseudotuberculosis and Arcanobacterium pyogenes,three kinds of bacterias were 12.5%,3.75% and 7.5%,respectively.Three pathogenic bacterias of lymphadenitis in goat were isolated successfully and the multiplex PCR detection method was detected in this study,it laid a foundation for the research of the prevention and control and the pathogenesis for lymphadenitis in goat.