Purpose: To develop a real-time PCR method for detecting the “top six” non-O157 STEC serotypes, and to validate its performance on artificially inoculated beef trim.
Methods: Real-time PCR primers and probes specific for the non-O157 STEC were designed and the PCR conditions were optimized. About 1 to 8 CFU of each serotype (O26, O45, O103, O111, O121 and O145) were spiked into 375 g of beef trim, stored overnight at 4°C, and then enriched with mTSB + 8 ug/ml vancomycin. Sample aliquots were taken after 12 h incubation at 42°C, and analyzed using real-time PCR. Results were confirmed using the USDA-FSIS MLG 5B.01 method.
Results: A multiplex real-time PCR assay for screening and detecting non-O157 STEC was developed and validated, using an intra-laboratory validation procedure based on the USDA FSIS MLG 5B.01 method. The detection limit of the real-time PCR method was 104 cells/ml and was determined by using cell dilutions in triplicate assays. The performance of the assay was assessed by using on 20 replicates of artificially inoculated beef samples and compared with the MLG 5B.01 method. The real-time PCR method gave 100% concordance with the MLG 5B.01 method. The developed real-time PCR assay detected non-O157 STEC at a level of ~1 to 8 CFU per 375 g beef samples after 12 h enrichment in modified tryptic soy broth (mTSB) supplemented with vancomycin.
Significance: This real-time PCR assay in combination with the modified enrichment medium effectively detects non-O157 STEC from spiked meat samples at a shorter incubation time than the USDA-FSIS MLG 5B.01 method.