A multiplex polymerase chain reaction assay for rapid detection and identification of Escherichia coli O157:H7 in foods and bovine feces
A multiplex polymerase chain reaction assay for rapid detection and identification of Escherichia coli O157:H7 in foods and bovine feces
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DOI：
10.4315/0362-028x-63.8.1032
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发表时间：
2000-08-01
影响因子：
2
通讯作者：
Pepe, T
中科院分区：
文献类型：
--
作者：
Fratamico, PM;Bagi, LK;Pepe, T
为简化大肠杆菌O 157：H7的检测，鉴定其H血清型和滋贺毒素类型，设计了一种多重PCR方法。质粒编码的溶血素基因（hly（933））、染色体鞭毛（fliC（h7）; H7血清群的鞭毛结构基因）、滋贺毒素（stx（1）、stx（2））和附着和清除（eaeA）基因的引物用于多重PCR，以共扩增肠出血性大肠杆菌的相应DNA序列。大肠杆菌O 157：H7。在PCR之前，对人工接种不同水平的大肠杆菌O 157：H7菌株933的碎牛肉、蓝纹奶酪、贻贝、苜蓿芽和牛粪的富集培养物进行简单的DNA提取步骤，并通过琼脂糖凝胶电泳分析所得扩增产物。检测灵敏度小于或等于1 CFU/g食物或牛粪（初始接种水平），可在24 h内获得结果。接种后立即进行富集培养的碎牛肉样品和富集前冷冻或冷藏的样品获得了类似的检测水平。多重PCR检测E.大肠杆菌O 157：H7，可将确认分离株所需的时间缩短3至4天。
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.