Development of a Rapid Microarray-Based DNA Subtyping Assay for the Alleles of Shiga Toxins 1 and 2 of Escherichia coli

Abstract

In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise.

Shiga Toxin-Producing Escherichia coli Infection in Jönköping County, Sweden: Occurrence and Molecular Characteristics in Correlation With Clinical Symptoms and Duration of stx Shedding

“…All STEC isolates were further subjected to microarray based assays with the E. coli SeroGenoTyping AS-1 Kit, ShigaToxType AS-2 Kit (Alere Technologies GmbH, Germany) to determine the serogenotype (O:H) (Geue et al, 2014a ) and stx allele/subtype (Scheutz et al, 2012 ; Geue et al, 2014b ) according to manufacturer's instructions ( http://alere-technologies.com/en/products/lab-solutions/e-coli/e-coli-serogenotyping-kit.html , http://alere-technologies.com/en/products/lab-solutions/shigatoxin.html ). Detection of eae and ehxA was done by PCR (Bai et al, 2016 ).…”

Genetic makeup of Shiga toxin-producing Escherichia coli in relation to clinical symptoms and duration of shedding: a microarray analysis of isolates from Swedish children

“…The three different microarray-based assays performed for each isolate included the determination of the serogenotype and the stx allele/subtype [12, 22], as well as the detection of antibiotic resistance genes, and a large number of other virulence genes [22, 25, 26]. …”

“…This information can also be used to assign isolates to different lineages and determine the genetic relationship between them, comparable with analysis of SNPs mentioned above and to discriminate between different stx subtypes [21, 22]. …”

The Changing Role of the Clinical Microbiology Laboratory in Defining Resistance in Gram-negatives

“…113,[122][123][124] This platform was evaluated against a panel of 117 Gram-negative isolates, showing an overall 100% concordance rate with the PCR method. 125 Nanosphere Technology (Northbrook, IL) has developed a promising automated microarray (Verigene Nucleic Acid Test) for detecting bacterial species and resistance genes within 2 hours.…”