Abstract：
Prototheca bovis is an algal emerging pathogen in dairy farms causing refractory protothecal mastitis with increasing incidence worldwide and significant economic impact. P. bovis infects cows throughout the lactation cycle, including dry periods, and can persist in the udder and the environment for a long time. Since P. bovis does not respond to treatments with antibiotics, the suggested sanitary measure to restrict the spread is culling infected animals. A point-of-care test for early detection of the causative agent is critically needed to guide farm management and the appropriate treatment of mastitis. Loop-mediated isothermal amplification (LAMP) is a highly specific molecular method, time-saving, cost-effective and easy to perform in limited settings. This study aimed to develop a LAMP assay for P. bovis detection directly from milk samples; it was employed in conjunction with a commercial DNA extraction kit which was previously used to extract DNA from milk specimens containing microbes. The LAMP assay detected P. bovis DNA within 1 h in milk samples spiked with P. bovis at a concentration of 50 cells/μL, enabling on-farm disease monitoring and decision-making based on a reliable diagnosis. The LAMP method will contribute to the accurate and rapid identification of P. bovis in asymptomatic or recurrent mastitis cases and consequently aid the implementation of targeted control measures and the reduction of losses in milk production.
摘要：
原藻牛是奶牛场中的藻类新兴病原体，可引起难治性原壳性乳腺炎，在全球范围内发病率增加并产生重大经济影响。牛分枝杆菌在整个泌乳周期中感染奶牛，包括干旱时期，并且可以在乳房和环境中长时间坚持。由于牛分枝杆菌对抗生素治疗没有反应，建议的限制传播的卫生措施是剔除受感染的动物。迫切需要对病原体进行早期检测的即时测试，以指导农场管理和乳腺炎的适当治疗。环介导等温扩增(LAMP)是一种高度特异的分子扩增方法,节省时间,具有成本效益和易于执行在有限的设置。这项研究旨在开发直接从牛奶样品中检测牛毕赤酵母的LAMP测定法；它与以前用于从含有微生物的牛奶样品中提取DNA的商业DNA提取试剂盒结合使用。LAMP测定法在1小时内检测到牛乳样品中以50细胞/μL的浓度掺入牛乳杆菌的牛乳杆菌DNA，能够在可靠诊断的基础上进行农场疾病监测和决策。LAMP方法将有助于在无症状或复发性乳腺炎病例中准确和快速地鉴定牛肝菌,并因此有助于实施有针对性的控制措施和减少产奶量损失。
