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.