Purpose: The purpose was to evaluate specificity and limit of detection of the PCR assays for the various gene targets, and to compare the performances to the USDA/FSIS 5B.01 for raw ground beef and raw beef trim after enrichment.
Methods: After enrichment of 375 g raw ground and beef trim in 1.5 l BPW for 10 h at 41.5°C, bacterial DNA was extracted, then analyzed with a multiplex PCR assay targeting the genes stx, eae and specific genes of the Top7 serogroups. When stx, eae and at least 1 specific gene of the Top7 serogroups were co-detected, the software displayed an alert indicating a presumptive positive. The method was evaluated using 400 ground and 150 trim processed in 4 beef facilities and compared to the USDA-FSIS MLG 5B.01 method. Presumptive positive samples were confirmed according to reference methods.
Results: Specificity of each PCR assay was demonstrated using 122 E. coli strains. The limit of detection of each PCR assay was 25 GU/PCR well, except for O145 (50 GU/PCR well), theoretically corresponding to 3E+04 and 6E+04 CFU/ml after enrichment, respectively. Beef samples artificially contaminated with O26, O103 or O157 showed 100% presence of all target genes at levels 10 - 2,500 CFU/PCR well. The multiplex and reference methods gave 3 and 7 presumptive positives in the uninoculated beef samples, respectively. Cultural confirmation yielded 3 Top7 STEC isolates, 2 of which were presumptively positive by multiplex. None of presumptive positives obtained by the reference method were confirmed.
Significance: The GeneDisc method may be useful for routine screening of beef samples for STEC Top7 serogroups with a time to result less than 12 hours.