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.

中文翻译：

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实时荧光定量 PCR 技术检测牛生乳中牛分枝杆菌和流产布鲁氏菌的验证

布鲁氏菌病和肺结核是对牛群经济影响巨大的疾病，在许多国家由政府计划控制。诊断技术的验证是其在这些疾病的官方控制计划中应用的基础。本研究的目的是验证实时聚合酶链反应 (qPCR)，用于检测人为污染的生奶样品中的牛分枝杆菌和流产布鲁氏菌。该技术通过分析灵敏度和特异性、重复性、内部再现性和稳健性测试进行评估。最初，测试了五种 DNA 提取方法，DNeasy Mericon Food Kit-Qiagen 和 Maxwell® 16 Tissue DNA Purification Kit-Promega 在所有测试的商业试剂盒中表现出最佳的分析特异性，并专门用于后续测试。在 qPCR 中获得的最低检测限是牛分枝杆菌 DNA 的 2.3 pg 和流产芽孢杆菌 DNA 的 20.7 fg。与稳健性相关的可重复性和再现性表明，所评估的方法可用作快速工具，用于巴西奶牛生奶中牛结核病和布鲁氏菌病的官方体内诊断。