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In this study, a duplex qPCR assay was developed for the needs of the Irish fish industry to screen for the two major food-borne pathogens of fish, Listeria monocytogenes and Escherichia coli O157:H7. The assay can claim positive or negative results for two pathogens in one go in only 20 h including 16 h universal pre-enrichment and compared to traditional ISO approved plate culture methods the labour and the cost involved in testing of one sample is reduced to minimum. The highly specific genomic areas targeted for PCR amplification in the assay are the hly gene for listeriolysin O (LLO) of L. monocytogenes and the stx gene for Shiga–like toxin expressed by E. coli O157:H7. The detection limit of the assay is consistent with the consumer protection limits of 1 pg genomic DNA or 1 CFU 25 g−1 fish meat (with enrichment) allowing the test to be considered as a substitute to standard plate culture methods.Significance and Impact of the Study: The study highlights a novel duplex qPCR for Listeria monocytogenes and Escherichia coli O157:H7 that could be used as an alternative to plate-based ISO or singleplex PCR methods while minimizing the costs. The assay uses rapid DNA extraction methods and locked nucleic acid probes. Sensitivity and specificity are 100 and 98·95% respectively. The potential for quantitative rage of the assay is 108–101 CFU ml−1.