牛乳中产气荚膜梭菌α毒素和金黄色葡萄球菌肠毒素A的双重PCR检测法  

Detection of Clostridium perfringens α-toxin and Staphylococcus aureus Enterotoxin A in Dairy with Double PCR Technique

作　　者：张曼[1,2] 王晶钰[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]杨凌职业技术学院动物工程系,陕西杨凌712100

出　　处：《西北农业学报》2010年第9期29-33,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家"十一五"科技支撑计划奶业专项(2006BAD4A11);陕西省"13115"科技创新工程重大科技专项(2008ZDKG-05)

摘　　要：建立牛奶乳样中产气荚膜梭菌α毒素和金黄色葡萄球菌肠毒素A的双重PCR检测方法。根据产气荚膜梭菌α毒素和金黄色葡萄球菌肠毒素A基因序列分别设计了2对特异性引物,分别从产气荚膜梭菌和金黄色葡萄球菌参考菌株的肉汤培养物中提取DNA,进行PCR扩增,并进行特异性、敏感性试验和临床样品检测。经过PCR反应条件的优化,建立了产气荚膜梭菌α毒素和金黄色葡萄球菌肠毒素A的双重PCR检测方法,均扩出了相应的目的条带。该方法将参考菌株的肉汤培养物稀释1 000倍后仍可扩出目的条带,且对大肠埃希菌等的PCR检测为阴性。对临床采集的50份乳品样本分别用细菌分离培养法和PCR方法进行检测,两种方法的符合率达90%以上,但双重PCR检测方法具有特异性强和敏感性高的特点。The study was to establish a duplicate PCR detection method for Clostridium perfringens α-toxin and Staphylococcus aureus enterotoxin A.in milk and to guarantee the safety of dairy products.According to gene sequences of Clostridium perfringens α-toxin and Staphylococcus aureus enterotoxin A,two pairs of specific primers were designed.The DNA templates were extracted respectively from the cultured bacterial liquids of Clostridium perfringens and Staphylococcus aureus and used for DNA amplification.The specificity and sensibility of the method were also detected,and some clinical samples were checked in this way.The result showeds that,based on the optimized reaction conditions of PCR,samples were processed DNA amplification for Clostridium perfringens and Staphylococcus aureus.The matching prospective bands were obtained in both bacteria.But this reaction system could not amplify products in other bacterium like Bacillus coli,which indicated this method had specificity.Even for the 1000-times-diluted bacterial liquids of Clostridium perfringens and Staphylococcus aureus,this method could also amplify the target gene fragment,which meant its high sensitive.This PCR method was also compared with routine bacteriology culture method,and the coincidence rate of these two methods was above 90%.In conclusion,a duplicate PCR detection method for Clostridium perfringens α-toxin and Staphylococcus aureus enterotoxin A had been established and it had high sensitivity and specificity.

关 键 词：产气荚膜梭菌 Α毒素 金黄色葡萄球菌 肠毒素A PCR检测 

分 类 号：S852.61[农业科学—基础兽医学]