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Comparison of an internally-controlled real-time PCR assay with the current plate-based assay for the detection of Bacillus sensu lato contaminants in gelatine.A comprehensive TaqMan® probe was designed allowing the real-time PCR assay to be fully inclusive for the gelatine-contaminating Bacillus s.l. species. An internal amplification control was implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (Kappa value = 0·94) was observed between the performance of the real-time PCR and the current plate-based method on naturally contaminated gelatines (n = 162). Relative accuracy, relative sensitivity and relative specificity were 97·5%.The real-time PCR assay is an adequate alternative of the current plate-based assay.The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h. The gelatine-producing industry can ensure gelatine quality in a much faster way.