Detection and quantification of viable Bacillus cereus in food by RT-qPCR.

摘要

A reverse-transcription real-time PCR (RT-qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C (pc-plc) mRNA, was developed for the specific detection and quantification of viable Bacillus cereus. Initial experiments focused on evaluating the performance of various RNA extraction kits and optimizing the DNase I digestion. After optimization, RNA from B. cereus was isolated, and following DNase treatment, the RNA was amplified by RT-qPCR. The assay was used to construct a calibration curve from purified RNA of B. cereus CECT 148T, and it had a wide quantification range of 5 log units. The detection limit was 30 CFU per reaction and the efficiency 0.88. When the developed methodology was applied in artificially contaminated liquid egg, the detection limit was found to be 850 CFU per reaction or 1.1 x 104 CFU per mL of food sample without an enrichment step. To the best of our knowledge, this is the first time that an assay for the detection and quantification of viable B. cereus in food has been described.