Real-time molecular beacon NASBA reveals hblC expression from Bacillus spp. in milk

Nucleic acid sequence-based amplification (NASBA) was applied in combination with a fluorescein-conjugated molecular beacon specific for a sequence flanked by transcript-specific primers in order to monitor hblC enterotoxin gene expression in real-time from milk separately contaminated with Bacillus amyloliquefaciens, Bacillus cereus, and Bacillus circulans. Maximal enterotoxin expression was noted following 16, 15, and 16h, respectively, when grown in artificially contaminated nonfat dried milk incubated aerobically at 32°C, corresponding to 1.6×10⁵, 5×10⁷, and 9.8×10⁴cfu/ml, for B. amyloliquefaciens, B. cereus, and B. circulans, respectively. This RNA amplification assay allows for simultaneous detection and confirmation of target transcripts in a closed tube format and may be performed in a high DNA background. The development of a rapid, sensitive, real-time method to quantitate the expression of virulence genes in pathogenic spore-formers is useful in shelf life determination of foods and other quality assurance measures.