Development of a Real-Time PCR assay for the specific detection of Brochothrix thermosphacta in fresh and spoiled raw meat.

Brochothrix thermosphacta is a psychrotrophic species commonly involved in the spoilage of meat and often recognized as the dominant organism causing off-flavours. The knowledge of the genera/species affecting meat spoilage is necessary to define a successful method for food preservation. The aim of this study was to develop a Real-Time (RTi-) PCR method for the species-specific detection of B. thermosphacta and to evaluate a RTi-PCR approach for its enumeration in fresh and spoiled beef, avoiding the culturing steps. The specificity of the primers designed on the basis of the 16S rRNA gene sequences of B. thermosphacta was tested using the DNA extracted from strains belonging to bacterial species usually associated with meat. The RTi-PCR assay allowed a species-specific detection of B. thermosphacta and no amplification signals were retrieved using DNA from the other species under the conditions used. Three different standard curves were constructed by using broth culture, a meat extract and meat samples containing different concentrations of B. thermosphacta. The standard using artificially contaminated meat samples was chosen because of its closeness to an authentic contamination case. The standard curve was linear in the range from 2.2 x 10(2) to 2.3 x 10(7)CFU/g; the reaction efficiency was 1.11. The RTi-PCR assay was then applied to enumerate B. thermosphacta in 20 fresh and spoiled beef samples and the results were compared to those obtained by plating onto selective medium for B. thermosphacta. A comparison between the two methods reported a general underestimation (from 0.5 to 2 Log CFU/g) of the microbial loads by RTi-PCR. Except for a few cases, the statistical analysis showed significant differences between viable counts and RTi-PCR data. The identification of B. thermosphacta by the RTi-PCR method developed in this study is certainly simple and fast and can be useful for its reliable detection in meat samples. However, considering the level of underestimation reached in most of the samples analyzed, the RTi-PCR method can be recommended only to approximately predict the contamination level of B. thermosphacta in meat.