Single-step method for rapid detection of Brucella spp. in soft cheese by gene-specific polymerase chain reaction

Brucellosis can be transmitted to man by direct contact with infected animals or through contaminated meat, milk and dairy products (Nicoletti, 1989). The analysis of Brucella spp. is carried out in the laboratory by microbiological or serological assays (Alton et al. 1988). The first are more specific but are also time-consuming and expose the analyst to the risk of infection (López-Merino, 1991). However, the latter can result in false positives owing to cross reactivity with other Gram-negative bacteria (Diaz-Aparicio et al. 1994). Because of these limitations, the amplification in vitro of specific DNA regions by the polymerase chain reaction (PCR) could represent a powerful tool for rapid and specific diagnostic analysis. In recent years, several PCR methods have been developed to amplify specific DNA sequences of Brucella strains (Herman & de Ridder, 1992; Romero et al. 1995; Valentino et al. 1997). In addition, direct analysis of Brucella in contaminated abortive tissues (Fekete et al. 1992), milk and blood (Leal-Klevezas et al. 1995; Rijpens et al. 1996) has been reported.In this paper we describe a method for gene-specific PCR amplification of a 443 base pair (bp) fragment of Brucella DNA that belongs to a gene encoding for a 31 kDa outer membrane protein. This protein (BCSP-31) is a membrane antigen characteristic of the Brucella genus (Mayfield et al. 1988). The PCR method was developed for the analysis of soft cheeses. We focused our attention on Mozzarella, Pecorino and ricotta samples, because such products are not subjected to the natural microbial autopurification process of maturing. They are widely consumed in Italy and a relationship between infected foods and the areas where brucellosis is a human zoonosis is a possibility.The analysis was performed without purification of DNA from bacteria. Indeed, after homogenization, the sample was subjected to thermal shock by freeze–thaw cycles that lysed bacteria and solubilized nucleic acids for subsequent PCR amplification. Amplified DNA fragments were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Several brands of soft cheeses and ricotta contaminated at different levels with Brucella cells were analysed by our procedure to evaluate the detection sensitivity and the repeatability of the method.