Development of a real‐time PCR assay with an internal amplification control for the screening of Shiga toxin‐producing Escherichia coli in foods

Aims:  To develop and evaluate a real-time PCR assay incorporating an internal amplification control (IAC) suitable for the screening of Shiga toxin (Stx)-producing Escherichia coli (STEC) in foods. Methods and Results:  A competitive IAC was constructed and included in an stx-specific real-time PCR assay. Coupled to 18-h enrichment and automated DNA extraction, the assay could reliably detect the presence of STEC in minced meats inoculated at 10 CFU per 25 g. Its performance was evaluated on 415 minced beef and 112 raw milk cheese samples and compared with that of a PCR-ELISA method. Fifty-three minced meats and 31 cheeses were found stx-positive, giving 98·3% and 93·75% concordance, respectively, with the PCR-ELISA reference method. Conclusions:  A highly sensitive stx-specific real-time PCR method including an IAC was developed, facilitating monitoring of false-negative results due to PCR inhibitors. Significance and Impact of the Study:  Combined with automated DNA extraction, the stx-IAC real-time PCR assay represents a suitable method for rapid screening of STEC in foods.