炭疽芽孢杆菌PCR鉴定方法的建立  

Identification of Bacillus anthracis strain by PCR method

作　　者：梁冰[1,2] 祝令伟[2] 纪雪[2] 周伟[2] 孙洋[2] 刘军[2] 郭学军[2] 冯书章[1,2] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国兽医学报》2016年第8期1363-1366,1370,共5页Chinese Journal of Veterinary Science

基　　金：农业部<动物疫情监测与防治(兽医)>资助项目(271);国家"863"计划资助项目(2012AA222006);吉林省青年科研基金资助项目(20150520130JH)

摘　　要：根据炭疽芽孢杆菌3种特异性毒力基因的序列，包括保护性抗原基因（pag）、荚膜基N（caP）和S-层蛋白基因（sap），建立多重PCR鉴定方法。该方法检测11株炭疽芽孢杆菌均为阳性，检测29株非炭疽的其他需氧芽孢杆菌属菌株均为阴性。该方法检测模拟血液样品的灵敏度是2×106 CFUjmL（每1mL血液样品最低舍有2×10s细菌），而样品增菌后的检测灵敏度是2×102CFU／mL。制备加入炭疽疫苗菌株芽孢的模拟土壤样品，经增菌培养后，PCR检测灵敏度为3×104芽孢／g。另外，还设计了炭疽芽孢外壁结构蛋白基因BclA的鉴定引物，利用该引物的PCR扩增片段长度以及测序结果。能够有效区分Ⅱ号炭疽疫苗菌株和临床分离菌株，本研究能够为炭疽临床诊断和环境监测工作提供技术支持。The multiplex PCR method was developed according to the three specific virulence genes of Bacillus anthracis ,including the protective antigen gene （pag）,capsule gene （cap） and S-layer protein gene （sap）. All the 11 Bacillus anthracis strains were identified as positive result by the PCR assay,and the other 29 Bacillus strains were identified as negative result. DNA of the simula- ted blood specimens were extracted and detected by the PCR assay, the detection limit was 2×106 CFU/mL （the lowest 2 × 106 Bacillus anthracis strains per 1 mL blood specimen） ; after en- richment culture of bacteria,the detection limit is 200 CFU/mL. The simulated soil samples were also prepared and detected by the PCR assay,after enrichment culture,the detection limit is 3× 104 spores/g in soil. In addition,the PCR primers of spore surface gly signed,which can effectively distinguish the Bacillus anthracis Ⅱ lares. coprotein gene （BclA） were de vaccine strains from clinical isolates.

关 键 词：炭疽芽孢杆菌 PCR 血液 土壤 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]