Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Rome, Italy.
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for paratuberculosis, Podenzano, Italy.
Microbiologyopen. 2019 Oct;8(10):e875. doi: 10.1002/mbo3.875. Epub 2019 Aug 17.
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 (2.6 × 10 cfu/ml) and 4.08 (26.7 cfu/ml) IS900 target copies and a LOD between 5.3 × 10 (4.1 × 10 cfu/ml) and 53 (4.1 × 10 cfu/ml) IS900 target copies, respectively.
本研究旨在开发和验证不同的创新 DNA 提取方法，以从牛和水牛初乳中检测分枝杆菌 avium subsp. 副结核杆菌（MAP）DNA。副结核病是一种由 MAP 引起的慢性炎症感染，可感染家养和野生动物，尤其是反刍动物。疾病的主要传播途径是粪便，但 MAP 也可以在牛奶和初乳中排出。2015 年，意大利卫生部发布了 MAP 的自愿控制计划，以便对牛和水牛农场进行基于风险的认证。除了年度诊断筛查和动物临床监测外，该计划还包括采用生物安全和管理措施，逐步减轻 MAP 的发病率。为了实现这一目标，至关重要的是要确保用于检测牛和水牛牛奶和初乳中 MAP 存在的方法的准确性，以：（1）支持“安全初乳农场银行”的建立，从而防止主要的场内 MAP 传播途径，（2）允许出口产品的 MAP 无认证。为了实现这些目标，从文献中确定了七种不同的 DNA 提取方案，其中三种方法在采用评估程序后最终被选中，该程序旨在评估 DNA 提取效率、DNA 纯度和 DNA 扩增的适应性：NucleoSpin 食品试剂盒（美吉）、NucleoSpin 食品试剂盒（美吉）与磁性珠结合，以及 QIAamp Cador 病原体迷你试剂盒（QIAGEN）。特别是，NucleoSpin 食品试剂盒（美吉）和 QIAamp Cador 病原体迷你试剂盒（QIAGEN）在牛和水牛初乳上进行了测试，显示出 4×10（2.6×10 cfu/ml）和 4.08（26.7 cfu/ml）之间的 LOD 以及 5.3×10（4.1×10 cfu/ml）和 53（4.1×10 cfu/ml）之间的 LOD 目标拷贝的 IS900 。
