Insertion/deletion (Indel) Based Approach for the Detection of Escherichia coli O157:H7 in Freshwater Environments

Abstract

<p>Though pathogenic strains represent a small portion of the total variety of existing <em>Escherichia coli </em>strains, they contribute extensively to human morbidity and mortality. Disease outbreaks caused by enterohaemorrhagic <em>E. coli</em> of the serotype O157:H7 and the “Big Six” serotypes (i.e., O26, O45, O103, O111, O121 and O145) have driven the development of assays for pathogen detection. From culture-based assays requiring several days for confirmation of target organisms, to quantitative PCR (qPCR) tests that provide pathogen identification in several hours’ time, the sensitivity, specificity and speed of bacterial diagnostics have seen improvements that increased the efficacy of assays used to detect pathogens at clinically relevant levels. One relatively unexplored field of diagnostics is the use of conserved signature insertion/deletions (CSIs) as stable genetic markers for pathogen detection. This thesis presents two qPCR assays that target an <em>E. coli</em> O157:H7-specific insertion in a CSI. In a more preliminary study, an EvaGreen-based qPCR assay was developed that had a detection limit of 16 <em>E. coli</em> O157:H7 genome equivalents. An improved format of the O157:H7-specific CSI assay, using TaqMan probes, was later established. TaqMan probes are sequence-specific, while DNA-intercalating EvaGreen dye is sequence-independent. Though the TaqMan probe-based assay had a higher detection limit of 100 genome equivalents, the assay maintained detection sensitivity in presence of genetically similar (<em>E. coli</em> K-12) and dissimilar (fish sperm) DNA in excess amounts (1000-fold and 800-fold excess of target DNA, respectively), demonstrating its potential for pathogen detection in environmental samples where the presence of background flora may influence detection. These assays thus represent an exploration into the use of CSIs as diagnostic tools. This thesis also provides a guide for future developments of pathogen detection using CSIs, such as those that may be present in toxigenic species of Cyanobacteria and human pathogens, including <em>Vibrio</em> and <em>Campylobacter</em>.</p>