弗劳地枸橼酸杆菌分离鉴定及其三重PCR 检测方法的建立  

Isolation and Identification of Citrobacter freundii and development of a Triplex PCR detection method

作　　者：张楠驰 王利[1,2] ZHANG Nan-chi;WANG Li(Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization,Ministry of Education,Southwest Minzu University,Chengdu 610041,China;Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province,Chengdu 610041,China)

机构地区：[1]西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都610041 [2]青藏高原动物遗传资源保护与利用四川省重点实验室,四川成都610041

出　　处：《中国预防兽医学报》2020年第7期669-674,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：四川省科技支撑项目(2016NZ0044)。

摘　　要：为鉴定导致鲫鱼嘴部溃烂、肛门红肿、腹部有出血点症状的病原菌,并建立一种快速检测该病原菌的方法,本研究采用形态学、生理生化特性分析及16S rDNA序列分析方法从患病鲫鱼中分离鉴定病原菌株,并进行了动物回归实验。结果显示,从患病鲫鱼中分离菌株为弗劳地枸橼酸杆菌(Citrobacter freundii),命名为fsznc-08,该菌对鲫鱼具有致病性。针对该菌GalF基因、petA基因和FliC基因设计了3条特异性引物,通过对反应体系和条件的优化,建立了检测该菌的三重PCR方法,结果显示,三重PCR方法可准确扩增出弗劳地枸橼酸杆菌GalF、petA和FliC 3个目的基因,而其它菌株均未扩增出目的基因,特异性良好;该方法对该菌DNA最低检出限为6.09×101拷贝/μL。利用该方法检测人工感染患病鱼样品的阳性率约为93.33%,与单一PCR方法的检测率一致,但特异性和敏感性均高于单一PCR。本实验建立的弗劳地枸橼酸杆菌三重PCR检测法具有特异性强、敏感性高、操作简单、成本低的优点,为临床弗劳地枸橼酸杆菌的检测提供可行方法。To identify the pathogenic bacterium that caused mouth ulceration,swelling and abdominal bleeding of Carassius auratus and establish a rapid detection method.In this study,a pathogenic bacterium was isolated from Carassius auratus,and identified by morphological,physiology biochemistry characteristics and 16S rDNA sequence analysis.Carassius auratus were inoculated with the bacterium as an animal regress experiment.The result showed that a Citrobacter freundii strain was isolated from Carassius auratus and named as fsznc-08.This strain was pathogenic to Carassius auratus.Three specific primers were designed for GalF,petA and FliC genes.A triplex PCR method was established through optimizing the reaction system and conditions.The result showed that GalF,petA and FliC genes of Citrobacter freundii could be amplified by triplex PCR method,while other strains did not amplified the target genes.The detection limits for the DNA of Citrobacter freundii was 6.09×101 copies/μL.The detectable rate of Citrobacter freundii in artificially infected fish was about 93.33%using the triplex PCR,which was the same as the single PCR.But the triplex PCR was more specific and sensitive than single PCR.The established triplex PCR detection method for Citrobacter freundii in this study was specific,high sensitive,simple operation and low cost,which provided a credible method for the detection of Citrobacter freundii in clinical practice.

关 键 词：弗劳地枸橼酸杆菌 快速检测 多重PCR 分离鉴定 

分 类 号：S852.61[农业科学—基础兽医学]