Abstract
Molecular methods have emerged as the most reliable techniques to detect and characterize pathogenic Escherichia coli. These molecular techniques include conventional single analyte and multiplex PCR, PCR followed by microarray detection, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing. The choice of methods used depends upon the specific needs of the particular study. One versatile method involves detecting serogroup-specific markers by hybridization or binding to encoded microbeads in a suspension array. This molecular serotyping method has been developed and adopted for investigating E. coli outbreaks. The major advantages of this technique are the ability to simultaneously serotype E. coli and detect the presence of virulence and pathogenicity markers. Here, we describe the development of a family of multiplex molecular serotyping methods for Shiga toxin-producing E. coli, compare their performance to traditional serotyping methods, and discuss the cost-benefit balance of these methods in the context of various food safety objectives.
Highlights
Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology
The clinical manifestations of Shiga toxin-producing Escherichia coli (STEC) infections range from mild watery diarrhea to severe complications of hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and even death (Levine et al, 1987; Gyles, 2007)
A 7-plex immunoassay was developed by Clotilde et al (2013) to identify O serogroups O26, O45, O103, O111, O121, O145, and O157
Summary
Microbead-based suspension array technology has emerged as a standard method for simultaneously detecting multiple biological analytes from one sample and has enabled a wide variety of applications in life science research, clinical diagnostics, food safety, and biodefense (McBride et al, 2003; Toro et al, 2013; Sun et al, 2014; Di Cristanziano et al, 2015; Khalifian et al., 2015; Silbereisen et al, 2015; Zhang et al, 2015; Kong et al, 2016). To generate the assay reagents, magnetic microbeads were covalently coupled to capture Ab according to the instructions provided with the BioRad Amine Coupling kit (BioRad, Hercules, CA, USA), using an amount of Ab based on a preliminary microplate ELISA data (1–10 μg/mL). This reaction is a common two-step carbodiimide protocol with N-hydroxysulfosuccinimide. When we expanded the validation to include 161 environmental STEC strains our Luminex immunoassay missed only one strain of O157 In this latter experiment we compared performance of our assay to standard assays and a microbead-based PCR serotyping assay (Clotilde et al, 2015). We already have working immunoassays for Stx (Clotilde et al, 2011), and we plan to add intimin, a virulence factor involved in E. coli attachment
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