To improve differential diagnosis of Bacillus cereus group and minimize a risk of bacilli related disease, a multiplex quantitative real-time PCR assay for a one step detection and differentiation of B. cereus sensu lato and emetic B. cereus spores in milk was developed. A qPCR assay with primers and probe targeting gyrB (gyrase B) gene was successfully established and applied in a multiplex qPCR approach with previously published 16S rRNA and ces (specific part of the cereulide) qPCR assays. An internal amplification control (IAC) was included to meet diagnostic PCR requirements. The inclusivity and exclusivity of the assay were assessed using a panel of 81 strains, including 45 B. cereus group, 19 non-B. cereus group and 17 non-Bacillus strains. The limit of detection (LOD) for B. cereus in artificial inoculation experiments was 1.91 x 10(3) spores ml(-1) milk with a mean recovery rate of 81%. Good coefficient of determination was achieved between the number of B. cereus spores added and the qPCR derived number of B. cereus cell equivalents (R-2 = 0.973). The real-time qPCR assay is specific and sensitive, providing an efficient diagnostic and monitoring tool for the implementation in food and clinical diagnostic labs. (C) 2012 Elsevier Ltd. All rights reserved.