A multiplex polymerase chain reaction assay for rapid detection and identification of <i>Escherichia coli</i> O157:H7 in foods and bovine feces

作     者：Fratamico, PM Bagi, LK Pepe, T 

作者机构：USDA Agr Res Serv Eastern Reg Res Ctr Wyndmoor PA 19118 USA Univ Naples Federico II Dept Pathol Prophylaxis & Food Inspect I-80137 Naples Italy 

出 版 物：《JOURNAL OF FOOD PROTECTION》 (J. Food Protection)

年 卷 期：2000年第63卷第8期

页      面：1032-1037页

核心收录：

学科分类：0832[工学-食品科学与工程（可授工学、农学学位）] 08[工学] 0836[工学-生物工程] 

主　　题：双壳纲/微生物学 干酪/微生物学 DNA 细菌/分析 DNA 细菌/分离和提纯 电泳 琼脂凝胶/方法 大肠杆菌O157/遗传学 大肠杆菌O157/生长和发育 大肠杆菌O157/分离和提纯 粪便/微生物学 食品微生物学 意大利 肉/微生物学 紫苜蓿/微生物学 聚合酶链反应/方法 敏感性与特异性 血清分型/方法 贝类/微生物学 动物 牛 

摘      要：A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly(933)), and chromosomal flagella (fliC(h7);flagellar structural gene of H7 serogroup), Shiga toxins (stx(1), stx(2)), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E, coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was less than or equal to 1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.