Many of the current accredited methods for the molecular detection of Shiga toxin-producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods can inaccurately conclude the presence of E.coli containing both stx and eae because of the inability of the methods to determine if the two genes originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate if a droplet digital PCR (ddPCR)-based method that does not require DNA isolation could reliably identify the presence of an STEC containing eae in beef samples by confirming that both genes reside within the same cell, even when present in a mixed culture. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works without the need for DNA isolation by partitioning intact cells into emulsion droplets, where they are lysed, and subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparisons were made between the dd-Check STEC Solution, the BAX System Real-Time PCR STEC assay suite (Hygiena), and the iQ-Check STEC PCR detection kit (Bio-Rad) using 37 unique simulations of E. coli contamination in ground beef. While no single platform was consistently superior at detecting eae and stx across all pathogens tested, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of inaccurately identified samples when screening for E. coli with a stx+, eae+ genotype because it can identify the co-existence of multiple virulence genes within a cell even when in the presence of a mixed microbial population containing identical genes. Ultimately, incorporation of this system could result in substantial cost savings by reducing the expenses incurred when product samples are incorrectly classified as containing E. coli with a stx+, eae+ genotype.

中文翻译：

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使用液滴数字PCR检测牛肉产品中产生志贺毒素的大肠杆菌（STEC）。

食品中产生志贺毒素的大肠杆菌（STEC）分子检测的许多当前认可的方法都依赖于基于PCR的筛查方法，用于病原体特异性遗传标记stx和eae。不幸的是，由于这些方法无法确定两个基因是否起源于单一生物体而不是生物体混合物，因此这些方法不能准确地推断出同时含有stx和eae的大肠杆菌的存在。这项研究旨在评估不需要DNA分离的基于液滴数字PCR（ddPCR）的方法是否可以通过确认两个基因都位于同一细胞内（即使存在）来可靠地鉴定牛肉样品中是否含有STEC。在混合文化中。本研究中使用的ddPCR系统，dd-Check STEC Solution（Bio-Rad），通过将完整的细胞分配到乳状液小滴中进行裂解，然后进行多重终点PCR，无需DNA分离即可工作。这使测定法能够区分单个生物同时包含stx和eae的样品与stx和eae驻留在不同生物中的样品。dd-Check STEC解决方案，BAX系统实时PCR STEC分析套件（Hygiena）和iQ-Check STEC PCR检测试剂盒（Bio-Rad）进行了比较，使用了37种独特的地面大肠杆菌污染模拟方法牛肉。尽管没有一个单一平台能够在所有测试的病原体中始终能够出色地检测出eae和stx，但结果表明，用stx +筛查大肠杆菌时，dd-Check STEC Solution可以减少不准确鉴定的样品数量，eae +基因型，因为即使在存在包含相同基因的混合微生物种群的情况下，它也可以识别细胞内多种毒力基因的共存。最终，如果减少将产品样本错误地归类为含有具有stx +，eae +基因型大肠杆菌的产品样本，则合并该系统可以通过减少费用来节省大量成本。