Woods Travis A, Mendez Heather M, Ortega Sandy, Shi Xiaorong, Marx David, Bai Jianfa, Moxley Rodney A, Nagaraja T G, Graves Steven W, Deshpande Alina
Department of Chemical and Biological Engineering, University of New Mexico Albuquerque, NM, USA.
Department of Chemical and Biological Engineering, University of New MexicoAlbuquerque, NM, USA; The New Mexico ConsortiumLos Alamos, NM, USA.
Front Cell Infect Microbiol. 2016 Aug 31;6:92. doi: 10.3389/fcimb.2016.00092. eCollection 2016.
Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx 1 , stx 2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.
产志贺毒素大肠杆菌（STEC）菌株对健康构成严重威胁，约一半与STEC相关的食源性疾病归因于受污染的牛肉。我们开发了一种检测方法，能够使用多重寡核苷酸连接-聚合酶链反应（MOL-PCR）检测技术，以快速且多重的方式对样本进行筛查，以检测几种重要的与STEC相关的血清群（O26、O45、O103、O104、O111、O121、O145、O157）和三种主要毒力因子（eae、stx 1、stx 2）。该检测方法可检测独特的STEC DNA特征，旨在用于来自与牛肉生产相关的各种来源的样本，为目前使用的多重聚合酶链反应检测方法提供一种多重且高通量的补充方法。多重寡核苷酸连接-聚合酶链反应（MOL-PCR）是一种基于核酸的检测技术，它依靠流式细胞术/图像细胞术和多重微球阵列来检测目标病原体中存在的基于核酸的特征。当针对代表不同STEC血清群和毒素基因谱的DNA样本进行测试时，STEC MOL-PCR检测方法在所有设计的序列标记上提供了大于90%的分析特异性。本文描述了这种11重检测方法的开发及其验证结果。这种高度多重化、但更重要的是动态且适应性强的筛查检测方法允许在发现与公共卫生相关的其他特征时将其纳入。随着对STEC相关疾病对公共卫生影响的探索，单个样本将需要更多关于分类的信息；因此，该检测方法可作为复杂筛查系统的基础。
