Validation of real-time PCR technique for detection of Mycobacterium bovis and Brucella abortus in bovine raw milk.

Brucellosis and tuberculosis are diseases of great economic impact in cattle herds and are controlled by governmental programs in many countries. The validation of a diagnostic technique is fundamental for its application in official control programs of these diseases. The aim of the present study was to validate a polymerase chain reaction in real time (qPCR) for detection of Mycobacterium bovis and Brucella abortus in samples of artificially contaminated raw milk. The technique was evaluated using tests of analytical sensitivity and specificity, repeatability, internal reproducibility, and robustness. Initially, five DNA extraction methodologies were tested, and the DNeasy Mericon Food Kit-Qiagen and the Maxwell® 16 Tissue DNA Purification Kit-Promega presented the best analytical specificity of all the commercial kits tested and were used exclusively in subsequent tests. The lowest limits of detection obtained in the qPCR were 2.3 pg for M. bovis DNA and 20.7 fg for B. abortus DNA. The repeatability and reproducibility associated with the robustness indicate that the evaluated methods are applicable as rapid tools for the official in vivo diagnosis of bovine tuberculosis and brucellosis in raw milk from dairy herds in Brazil.