变性高效液相色谱检测沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌  

Identification of Salmonella,Campylobacter jejuni and Enterohemorrhagic E.coli by Denaturing High-performance Liquid Chromatography

作　　者：徐君怡[1] 曹际娟[1] 郑秋月[1] 刘淑艳[1] 徐杨[1] 

机构地区：[1]辽宁出入境检验检疫局,大连116015

出　　处：《生物技术通报》2009年第3期127-131,共5页Biotechnology Bulletin

基　　金：国家"十一五"科技支撑计划课题(2006BAK02A13)

摘　　要：应用多重PCR反应(multiplex PCR,mPCR)结合变性高效液相色谱(denaturing high-performance liquidchromatography,DHPLC)技术建立食品中沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的快速检测方法。以编码沙门氏菌的fimY基因、编码空肠弯曲菌的gyrA基因和编码肠出血性大肠杆菌O157:H7的rfbE基因为靶基因,选择3对引物,建立并优化了快速鉴别沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的多重PCR体系,扩增产物分别为284、159和499 bp,并验证了该多重PCR具有特异性。沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7标准菌株稀释成不同梯度,做灵敏度检测。试验结果表明该方法有很好的特异性,且灵敏度高,检测限可达到:沙门氏菌1.5 CFU/ml、空肠弯曲菌15 CFU/ml、肠出血性大肠杆菌O157:H7 15 CFU/ml。在随机采集的226份冷冻鸡肉类样品中,检出了7份样品为沙门氏菌阳性、10份为空肠弯曲菌阳性、1份为肠出血性大肠杆菌O157:H7阳性。研究建立的多重PCR-DHPLC方法可特异、灵敏地实现对沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的快速检测。A new molecular technique for the analysis of Salmonella, Campflobacter jejuni and enterohemorrhagic E.coli based on the separation of PCR-amphfied target fragments by denaturing high-performance liquid chromatography （DHPLC） was reported. A multiplex PCR （mPCR） assay was developed by using 3 sets of primers that specifically amphfy segments of the fimY,gyrA and rfbE genes,the PCR Products of which were 284,159 and 499 bp,respectively. Analysis of 22 strains demonstrated that this PCR system was specific. The detection limit of the mPCR was 1.5 CFU/ml for Salmonella, 15 CFU/ml for Campylobacter jejuni and 15 CFU/ml for enterohemorrhagic E.coli 0157：H7. 10 of Campylobacterjejuni and 1 of O157：H7 were detected in 226 chicken meat samples. These results indicated that the multiplex PCR-DHPLC assay can be used for specific and sensitive detection of salmonella, Campylobacter jejun and enterohemorrhagic E.cali O157：H7.

关 键 词：沙门氏菌 空肠弯曲菌 肠出血性大肠杆菌O157:H7 变性高效液相色谱 

分 类 号：Q93-3[生物学—微生物学]