Department of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Animal Research Center, Hokkaido Research Organization, Shintoku, Hokkaido 081-0038, Japan.
J Vet Med Sci. 2020 Jul 10;82(7):875-880. doi: 10.1292/jvms.19-0695. Epub 2020 May 22.
Polymerase chain reaction (PCR) is typically used for the early detection of mycoplasma in bovine milk; it requires 3 days to obtain results because of the necessary enrichment process. A more rapid, simple, and accurate detection method is required to directly detect the Mycoplasma bovis (M. bovis) gene in milk. In this study, we assess the utility of combining the following two methods to achieve this goal: the loop-mediated isothermal amplification (LAMP), which is more sensitive than PCR, and the procedure for ultra rapid extraction (PURE), which adsorbs and filters components that inhibit DNA amplification/detection. LAMP was examined using DNA extracts obtained by four methods. This showed that PURE had the highest sensitivity and specificity and that the combination of PURE and LAMP was able to detect M. bovis in milk. We then showed that the detection limit of M. bovis was 10 colony-forming units per milliliter of milk using the PURE-LAMP. Finally, the respective sensitivities of the PURE-LAMP and PCR were 57% and 86% for bulk tank milk, 89% and 74% for mature milk, 85% and 92% for colostrum/transitional milk, and 97% and 95% for mastitis milk. The specificity was 100% for all milk samples in both LAMP and PCR. We conclude that PCR was suitable for detecting mycoplasma in bulk tank milk and that the PURE-LAMP could detect mycoplasma within 2 hr and was also effective for mature and mastitis milk.
聚合酶链反应（PCR）通常用于牛初乳中支原体的早期检测；由于需要进行必要的富集过程，因此需要 3 天才能获得结果。需要一种更快速、简单和准确的检测方法来直接检测牛支原体（M. bovis）基因在牛奶中的存在。在本研究中，我们评估了以下两种方法相结合的效用：环介导等温扩增（LAMP），其比 PCR 更灵敏，以及超快速提取（PURE）程序，该程序吸附和过滤抑制 DNA 扩增/检测的成分。使用通过四种方法获得的 DNA 提取物来检查 LAMP。结果表明，PURE 具有最高的灵敏度和特异性，并且 PURE 和 LAMP 的组合能够检测牛奶中的 M. bovis。然后我们表明，使用 PURE-LAMP 可以检测到牛奶中每毫升 10 个菌落形成单位的 M. bovis。最后，PURE-LAMP 和 PCR 的灵敏度分别为 57%和 86%，用于批量奶，89%和 74%用于成熟奶，85%和 92%用于初乳/过渡奶，97%和 95%用于乳腺炎奶。在 LAMP 和 PCR 中，所有牛奶样本的特异性均为 100%。我们得出结论，PCR 适用于检测批量奶中的支原体，而 PURE-LAMP 可在 2 小时内检测到支原体，并且对成熟和乳腺炎奶也有效。
