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The aim of this study was to develop and validate different innovative DNA extraction methods to detect Mycobacterium avium subsp. paratuberculosis (MAP) DNA from bovine and buffalo colostrum. Paratuberculosis is a chronic inflammatory infection of domestic and wild animals, especially ruminants, caused by MAP. The primary route of disease transmission is feces, but MAP can also be excreted in milk and colostrum. In 2015, the Italian Ministry of Health has issued a voluntary control plan of MAP in order to allow risk-based certification of bovine and buffaloes farms. In addition to the annual diagnostic screening and to the clinical surveillance of animals the plan includes the adoption of biosecurity and management measures to progressively mitigate the incidence of MAP. To achieve this goal it is crucial to ensure the accuracy of the methods used to detect the presence of MAP in bovine and buffaloes milk and colostrum, in order to: (1) support a "safe colostrum farm-bank" set-up and thus prevent the main within-farm MAP transmission route and (2) to allow the MAP-free certification of milk products for export purposes. To achieve these goals, seven different DNA extraction protocols were identified from bibliography, out of which three methods were finally selected after the adoption of an evaluation procedure aimed at assessing the efficiency of extraction of DNA, the purity of DNA and the adaptability of the DNA amplification: NucleoSpin ® Food Kit (Macherey-Nagel), NucleoSpin ® Food Kit (Macherey-Nagel) combined with the magnetic beads, and QIAamp Cador Pathogen Mini kit (QIAGEN). In particular, the NucleoSpin ® Food Kit (Macherey-Nagel) and the QIAamp Cador Pathogen Mini kit (QIAGEN) were tested on bovine and buffalo colostrum, showing a LOD between 4 × 10 4 (2.6 × 10 6  cfu/ml) and 4.08 (26.7 cfu/ml) IS900 target copies and a LOD between 5.3 × 10 5 (4.1 × 10 6  cfu/ml) and 53 (4.1 × 10 3  cfu/ml) IS900 target copies, respectively., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)