|Burns, Frank - Dupont Qualicon|
|Bono, James - Jim|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/3/2010
Publication Date: 8/1/2010
Citation: Burns, F.R., Bono, J.L. 2010. Utilization of evolutionary model, bioinformatics and heuristics for development of a multiplex Escherichia coli O157:H7 PCR assay [abstract]. International Association for Food Protection. 2010:27.
Technical Abstract: Introduction: Escherichia coli O157:H7 is a devastating foodborne pathogen causing many foodborne outbreaks worldwide with significant morbidity and mortality. The plasticity of the E. coli O157:H7 genome, inconsistent expression of surface antigens, and sharing of genetic elements with other non-pathogenic bacteria (many of whom have yet to be characterized) complicates the development of a reliable and specific PCR assay for this organism. Purpose: The purpose was to develop a PCR-based assay for E. coli O157:H7 that would be inclusive of all E. coli O157:H7 isolates, including all known atypical isolates (e.g., rough, sorbitol-fermenting, telluride-sensitive isolates), while maintaining sufficient specificity for use as a reliable screening tool. Methods: The genetic evolutionary model for E. coli O157:H7 was used to broadly determine that specific targets from both the O55:H7 parental lineage, as well as the O157 encoding gene cluster (acquired as the last step in the evolution of this pathogen), should be employed. Bioinformatics and heuristic approaches were undertaken to identify multiple candidates from both of these lineage elements. Targets were then tested against over 250 E. coli O157:H7 isolates from both the DuPont and USDA, ARS, U.S. Meat Animal Research Center strain collections. In addition, these targets were tested against 350 ground beef enrichments to evaluate potential for non-specific crossreaction with genetically uncharacterized bacteria. Results: Of the eight targets evaluated, two failed at being completely inclusive, both missed isolates of the sorbitol-fermenting, telluride-sensitive lineage responsible for several European outbreaks. Of the remaining six targets, three had acceptably low rates of non-specific crossreactivity, two of these from the O157 gene cluster. The first target chosen had greater specificity utilizing two single nucleotide polymorphisms and was non-reactive with most O157-bearing bacteria that were not O157:H7. The second target chosen was from the O55:H7 parental strain and exploited de novo nucleotides flanking a truncated and disabled insertion sequence. Significance: An understanding of E. coli O157:H7 evolution, coupled with bioinformatics and heuristic approaches to dealing with largely uncharacterized background flora has allowed the development of a highly specific and inclusive multiplex PCR assay without the complications and failure modes introduced by upfront antibody-based selection.