Improved method for rapid DNA extraction of mastitis pathogens directly from milk
Improved method for rapid DNA extraction of mastitis pathogens directly from milk
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DOI：
10.3168/jds.s0022-0302(06)72080-x
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发表时间：
2006-01-01
影响因子：
3.5
通讯作者：
Luzzana, M
中科院分区：
文献类型：
--
作者：
Cremonesi, P;Castiglioni, B;Luzzana, M
有效控制牛乳腺炎需要灵敏、快速和特异性的检测，以检测和识别给乳制品行业造成重大损失的主要细菌。病原微生物的分子检测基于目标病原体的DNA扩增。因此，从病原菌中高效提取DNA是一个重要步骤。在这项研究中，我们旨在开发一种特异性、灵敏且快速的方法，直接从牛奶样品中发现的已知引起牛乳腺炎的主要革兰氏阳性菌（金黄色葡萄球菌、无乳链球菌、停乳链球菌和乳房链球菌）中提取 DNA。 DNA 提取方法基于离液剂硫氰酸胍的裂解和核酸酶灭活特性以及二氧化硅颗粒的核酸结合特性。开发了包含 6 个基本步骤（其中 3 个完成两次）的有效方案，并将其直接应用于牛奶样品。通过对目标细菌的物种特异性 DNA 序列进行 PCR 扩增来评估是否存在 PCR 抑制剂和 DNA 质量。我们实验中达到的灵敏度水平适用于无需样品富集的牛奶样品分析。
Efficient control against bovine mastitis requires sensitive, rapid, and specific tests to detect and identify the main bacteria that cause heavy losses to the dairy industry. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogen. Therefore, efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive, and rapid method to extract DNA directly from the main gram-positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis) found in milk samples. The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate, together with the nucleic acid-binding properties of the silica particles. An efficient protocol consisting of 6 basic steps (3 of which were done twice) was developed and applied directly to milk samples. Absence of PCR inhibitors and DNA quality were evaluated by PCR amplification of the species-specific DNA sequences of the target bacteria. The level of sensitivity achieved in our experiments is applicable to milk sample analysis without sample enrichment.