Development of real-time PCR assays for detection and quantification of Bacillus cereus group species: differentiation of B. weihenstephanensis and rhizoid B. pseudomycoides isolates from milk

Abstract

Quantitative real-time PCR (qRT-PCR) offers an alternative method for the detection of bacterial contamination in food. This method provides the quantitation and determination of the number of gene copies. In our study, we established an RT-PCR assay using the LightCycler system to detect and quantify the Bacillus cereus group species, which includes B. cereus, B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides, and B. pseudomycoides. A TaqMan assay was designed to detect a 285-bp fragment of the motB gene encoding the flagellar motor protein, which was specific for the detection of the B. cereus group species, excluding B. pseudomycoides, and the detection of a 217-bp gene fragment of a hypothetical protein specific only for B. pseudomycoides strains. Based on three hydrolysis probes (MotB-FAM-1, MotB-FAM-2, and Bpm-FAM-1), it was possible to differentiate B. weihenstephanensis from the B. cereus group species with nonrhizoid growth and B. pseudomycoides from the whole B. cereus group. The specificity of the assay was confirmed with 119 strains belonging to the Bacillus cereus group species and was performed against 27 other Bacillus and non-Bacillus bacteria. A detection limit was determined for each assay. The assays performed well not only with purified DNA but also with DNA extracted from milk samples artificially contaminated with bacteria that belong to the B. cereus group species. This technique represents an alternative approach to traditional culture methods for the differentiation of B. cereus group species and differentiates B. weihenstephanensis and B. pseudomycoides in one reaction.

FIG. 1.

Standard curve for determination of motB gene copy numbers using BCFomp2/BCRomp2 primers and MotB-FAM-1/MotB-FAM-2 probes. Crossing-point values were plotted against the log of the initial template DNA concentration. Plus or minus 1 standard deviation is indicated for each gene copy number. The average efficiency of every real time amplification was 2.0489 ± 0.078.

FIG. 2.

Standard curve for determination of gene copy numbers of B. pseudomycoides based on hypothetical gene using BpmF/BpmR2 primers and Bpm-FAM-1 probe. Crossing point values were plotted against the log of the initial template DNA concentration. Plus or minus 1 standard deviation is indicated for each gene copy number. The average efficiency of every real-time amplification was 1.95 ± 0.007.