An Optimized PCR Assay to Detect Escherichia Coli Harboring the astA Gene Encoding the Enteroaggregative E. coli...

Diarrheagenic Escherichia coli strains harboring the astA gene which encodes for the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) have been implicated in several foodborne outbreaks in Japan even in the absence of any other specific virulence marker of each pathovar. The polymerase chain reaction (PCR) assay is a critical tool used for detection and many astA specific primer sets have been developed though their specificity and sensitivity for astA detection directly from food samples have not been evaluated. Herein, four distinct PCR primer sets and three enzymes were evaluated in enriched food cultures to optimize astA detection. The Yamamoto & Echeverria PCR method yielded clear, easily interpretable results with high intensity of PCR product or no products. Combining this primer set with the Quick Taq HS DyeMix enzyme resulted in astA-specific amplicons without non-specific products from food cultures, indicating the superiority of this system in detecting astA in food samples. Furthermore, this primer set demonstrated the highest consistency with the E. coli harboring astA-isolation results. Subsequently, this system exhibited high specificity and sensitivity with a ≤5 log CFU/mL detection limit. These findings suggest that combining the Yamamoto & Echeverria primer set and the Quick Taq HS DyeMix offers an effective tool for detecting astA in food samples. We anticipate this PCR assay will enhance the detection and subsequent isolation of E. coli strains harboring astA from food products.