Detecção de células viáveis de Salmonella spp. e Staphylococcus aureus em queijo de coalho pela técnica de PCR em tempo real

Abstract

In many cases, milk and milk products are responsible to cause foodborne illness through transmission of microorganisms, like Salmonella spp. and Staphylococcus aureus. The contamination of these products can mainly occur due inefficient thermal processing or the lack of practices of hygiene and cleaning during the various stages of the production process, like occur during food handling or, even, after the thermal treatment. The rapid identification of pathogens in foods is extremely important, both for the quality assurance of products, such as in cases of outbreaks. Thus, the use of highly sensible and specific methods to detect food pathogens becomes indispensable. One of the techniques has been used to this is the Real Time PCR (qPCR), due its quickness and efficiency to identify pathogens in foods. Nevertheless, one of the major disadvantages of this technique is its inability to differentiate the DNA of viable and nonviable cells of microorganisms. To overcome this drawback, the ethidium bromide monoazide (EMA) can be used to detect viable cells only. EMA is a DNA intercalating dye that can enter selectively in cells with damaged membrane (considered dead) and bind covalently to DNA when exposed to halogen light, inhibiting its amplification during qPCR. So, the aim of this work was establish a protocol to detect in multiplex viable cells of Salmonella spp. and Staphylococcus aureus in pure cultures and Coalho cheese by use of EMA combined to Real-time PCR technique. The protocol established is efficient to identify viable cells of Salmonella spp., both in pure culture as for Coalho cheese. However, was observed that the differentiation between viable and nonviable cells of S. aureus by use of EMA is not efficient. Therefore, is not possible to detect viable cells of these pathogens in multiplex in pure cultures and in Coalho cheese. Moreover, it was observed that the established protocol, combining the qPCR technique and EMA, was able to detect viable cells concentrations of Salmonella typhimurium as low as 101 CFU/10g of Coalho cheese. However, it could only statistically differentiate the means of Cycle threshold (Ct) values in cells concentrations above to 103 UFC/10 g of cheese. The developed protocol is, therefore, an useful tool for food surveillance, since it provides rapid and specific identification of viable cells of Salmonella spp. in Coalho cheese.