100A-3 |
Enterotoxin gene expression in Bacillus cereus quantitatively ascertained using real-time multiplex molecular-beacon NASBA |
H. M. GORE, School of Biological Sciences, Louisiana Tech University, Ruston, LA 71272 and J. L. McKillip. NASBA (nucleic acid sequence-based amplification) is a method to isothermically amplify RNA sequences for studing gene expression without DNase treatment or blotting and hybridization. NASBA combined with molecular beacon probes reduces the time required to observe expression of specific genes in pathogens of interest. The organism used in this study to demonstrate the utility of the real-time assay is the dairy-associated pathogen Bacillus cereus. The objective of this study was to develop a rapid and sensitive method to monitor expression of two enterotoxin genes in separate operons from B. cereus during growth in milk. B. cereus was grown in skim milk under aerated conditions at 25oC for 8,12, and 16 hours. RNA was extracted and used in multiplex NASBA containing oligonucleotide primers and fluorecently-labeled molecular beacons specific for the hblC and nheA genes. Fluorescence was plotted in real-time and relative quantitation ascertained at each time point. Cell densities at 8,12, and 16 hours corresponded to 106, 108, and 109 CFU ml-1, respectively. Real-time fluorescence data, obtained following the 90 minute NASBA assay, indicated both hblC and nheA were maximally expressed at 12 hours. At all timepoints, detectable fluorescence emitted from the hblC-specific probe was higher than that of the nheA-specific beacon, suggesting the HBL operon in B. cereus is expressed at higher levels under the incubation conditions used for the artificially contaminated milk. Our data demonstrate the potential of combining real-time RNA amplification with molecular beacons to study enterotoxin gene expression in a model food system artificially contaminated with B. cereus. The sensitivity and speed of this assay are complemented by single tube amplification and confirmation offered by the sequence-specific molecular beacons. This technology holds promise in the food industry to screen products for growth and expression of virulence genes in target pathogens.
Session 100A, Food Microbiology: General II
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