Escherichia coli O157:H7 is an important food-borne pathogen and has ability to contaminate food, such as water, meat products, and milk. Moreover, E. coli O157:H7 is the main toxin-producing serotype which can produce Shiga toxin type 1 and type 2 and cause intestinal disease. The strong pathogenicity and lethality of Escherichia coli O157:H7 pose a serious threat to human. This study aims to develop a rapid and visual detection assay of E. coli for rfbE, stx1, and stx2 genes by crossing priming amplification (CPA). The limit of detection of CPA assay for rfbE, stx1, and stx2 genes was 3.20 fg/μl, 320 fg/μl, and 320 fg/μl in genomic DNA, while that of in artificially contaminated food samples was 10³ cfu/ml, 10⁵ cfu/ml, and 10⁵ cfu/ml, respectively, which was distinctly higher than that of PCR methods. And the specificity of CPA assay was tested by 22 different bacterial strains and except for E. coli O157:H7 ATCC43895 and other E. coli O157:H7 isolated from eggs, milk, and beef, all strains showed negative results. The visible detection assay was conducted by the addition of calcein in the reaction solutions. The CPA assay showed a successful detection of E. coli O157:H7 (Shiga toxin–producing and non-Shiga toxin–producing) within 60 min under 63 °C with high sensitivity and specificity. These results indicated that the CPA assays with calcein can provide an advanced method to achieve the rapid and visual detection of food-borne E. coli O157:H7.

中文翻译：

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通过交叉引物扩增（CPA）目测快速检测食品中的大肠杆菌O157：H7

大肠杆菌O157：H7是一种重要的食源性病原体，具有污染水，肉制品和牛奶等食物的能力。另外，大肠杆菌O157：H7是主要的毒素产生血清型，其可以产生1型和2型志贺毒素并引起肠道疾病。大肠杆菌O157：H7的强致病性和致死性对人类构成严重威胁。这项研究旨在通过交叉引物扩增（CPA）开发一种针对rfbE，stx1和stx2基因的大肠杆菌的快速直观检测方法。rfbE，stx1和stx2的CPA分析的检测限基因组DNA中的基因分别为3.20 fg /μl，320 fg /μl和320 fg /μl，而在人工污染的食品样品中，基因分别为10 ³ cfu / ml，10 ⁵ cfu / ml和10 ⁵ cfu / ml。 ，明显高于PCR方法。并且通过22种不同的细菌菌株测试了CPA测定的特异性，除了从鸡蛋，牛奶和牛肉中分离的大肠杆菌O157：H7 ATCC43895和其他大肠杆菌O157：H7以外，所有菌株均显示阴性结果。通过在反应溶液中加入钙黄绿素进行可见检测测定。CPA分析表明已成功检测到大肠杆菌O157：H7（产生志贺毒素和产生非志贺毒素）在63°C下60分钟内具有很高的灵敏度和特异性。这些结果表明，用钙黄绿素进行的CPA分析可以提供一种先进的方法，以实现对食源性大肠杆菌O157：H7的快速直观检测。