Detection and Quantification of Escherichia coli in Shellfish by Combined MPN-qPCR Method

Education Level

Undergraduate

Faculty Advisor(s)

Professor Galit Sharon

Academic Department(s)

Aquatic Diagnostic Laboratory, Center for Economic and Environmental Development, Roger Williams University and Coastal Ocean Vision, North Falmouth, MA.

Comments

This research was presented at the 2024 Rhode Island Summer Undergraduate Research Symposium, held on Friday, July 26, at the University of Rhode Island and supported by Roger Williams University.

Symposium Date

2024

Abstract

There is substantial need for the rapid and efficient detection and quantification of Escherichia coli and other fecal coliforms in shellfish intended for human consumption. Outbreaks of diarrhea, hemorrhagic colitis, and Hemolytic Uremic Syndrome (HUS) in humans have been associated with E. coli outbreaks, most commonly the O157:H7 strain due to its production of pathogenic Shiga-toxins. The current FDA- approved most probable number (MPN) method used for quantifying pathogenic E. coli in shellfish is outdated and can take up to four days to complete. In this project, two triplex quantitative Polymerase Chain Reaction (qPCR) assays were developed using previously published primers and probes. These assays are intended to be combined with the overnight enrichment from the standard FDA MPN method for the detection and quantification of the pathogenic O157:H7 E. coli strain. The assays were validated against known amounts of Shiga-toxin producing O157:H7 from cultures and confirmed to detect the strain. This streamlined qPCR-MPN method will be troubleshooted and validated with known amounts of O157:H7 E. coli through spiking experiments and later used to enumerate E. coli from summer shellfish collections. These qPCR assays will eventually be offered as part of the standard diagnostic methods in the Aquatic Diagnostic Laboratory at Roger Williams University.

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