Validation of larger test portion sizes for Escherichia coli Shiga toxin producer (STEC) detection by Immunomagnetic separation and Real Time PCR in meat and vegetables

Abstract

The aims of this study were (i) to evaluate the performance of the Assurance® GDS method combining immunomagnetic separation and Real Time PCR for STEC detection in pooled samples (up to 375g) of vegetables and meat and (ii) to compare its performances to that of the reference method ISO/TS-13136:2012 (25 g sample size) in artificially contaminated samples. The alternative Assurance® GDS method includes enrichment in proprietary broth at 41.5ºC for 10 h followed by primary screening of TOP 7 STEC containing eae, stx genes and O157:H7 markers using MPX-Top 7 (IMS+PCR), secondary screening for serogroup identification using MPX-ID/EHEC-ID (IMS+PCR). And finally, cultural confirmation from same IMS beads on two selective agars is set up. For reference method, the enrichment was performed in BPW at 41.5ºC for 18 h. A total of 120 samples of meat and vegetables, including 20 uninoculated and 100 samples spiked with stressed cells (<12 CFU/25 or 375g) of STEC, were analyzed using both methods. Our results showed that the Assurance® GDS method LOD50 ranged from 0.12 CFU/375 g to 0.522 CFU/375 g and the LOD95 from 0.518 CFU/375 g to 2.257 CFU/375 g. These data are similar to the LOD50 data of the reference method.