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Real-time PCR (qPCR) has been used for rapid identification of Salmonella Typhimurium and Staphylococcus aureus in dairy foods, but is unable to differentiate viable and unviable pathogens. Ethidium bromide monoazide (EMA), a DNA-intercalating agent, can detect only viable cells because selectively enter cells considered unviable and bind to their DNA, inhibiting its amplification during qPCR. The objective was to establish a protocol for detection of viable Salmonella Typhimurium and S. aureus, experimentally inoculated in coalho cheese, by the use of EMA combined with qPCR. The protocol was effective for the identification of viable Salmonella Typhimurium in coalho cheese but not for the S. aureus cells. Concentrations of viable Salmonella Typhimurium cells of 1 CFU/10 g of coalho cheese could be detected. The monoplex protocol enables the rapid and specific identification of viable Salmonella Typhimurium in coalho cheese, making it an alternative method for the quality and safety control of cheeses.

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