Development of a novel real-time PCR assay to detect intact astA-harboring Escherichia coli in food based on complete DNA sequence of astA gene variants.

Enteroaggregative Escherichia coli (EAEC) may produce an enterotoxin termed EAEC heat-stable enterotoxin 1 (EAST1), which is encoded by the astA gene. E. coli strains harboring astA but without virulence markers specific for other diarrheagenic E. coli pathotypes (EAST1EC), have been isolated from patients with diarrhea in foodborne outbreaks in Japan. Various nucleotide variants of astA have been reported, some of which are incomplete and non-functional. However, the astA variants carried by strains isolated from foodborne outbreaks have not yet been fully characterized. Therefore, we identified astA variants among various astA-harboring bacterial strains including EAST1EC isolated from 12 foodborne outbreaks. The majority of foodborne outbreak strains harbored multiple copies of intact astA variants, although EAST1EC from other origins harbored one of intact astA or incomplete and non-functional variants. To detect only intact astA-harboring E. coli in food, we designed a duplex real-time PCR targeting astA and 16S rRNA gene as an internal control (astArtPCR). The specificity of astArtPCR assay was tested along with four other PCR assays, and only the astArtPCR assay specifically detected intact astA variants. The astArtPCR when used in combination with enrichment had a detection limit of <2.6 log CFU/mL in food-enriched cultures. Overall, all E. coli strains harboring intact astA variants were positive for both astA and internal control with astArtPCR. The astArtPCR would be a useful tool for identifying the food related to foodborne outbreaks of EAST1EC and food surveillance.