布鲁氏菌可视化环介导等温扩增方法的建立及应用

Establishment and Application of Visual Loop-mediated Isothermal Amplification of Brucella spp.

摘要:

布鲁氏菌病（简称布病）是一种由布鲁氏菌引起的人畜共患疾病，影响了畜牧业的健康发展，甚至威胁到了人类的健康安全，造成了较严重的经济社会损失。因此，对布鲁氏菌病的防控十分必要。本研究主要基于布鲁氏菌保守基因Omp2a，利用在线网站设计3对引物，并优化其反应条件与反应体系，建立了一种检测布鲁氏菌的可视化LAMP方法。同时，将建立好的LAMP方法与聚合酶链式反应（PCR）检测方法相比较，并交由陕西省疾病预防控制中心进行实际样本检测。结果表明，基于羟基萘酚蓝（HNB）指示剂，LAMP扩增的结果可凭肉眼观察。建立的LAMP检测方法能准确区分布鲁氏菌与非布鲁氏菌，检测限为2.56×10-4 ng/μL。在实际样品检测中，LAMP检测方法相对于常规分离鉴定方法的敏感性和特异性均为100%。因此，本研究建立的LAMP检测布鲁氏菌方法具有特异性强、灵敏度高等优点，扩增45 min即可肉眼观察到检测结果，适用于现场和基层检测。

Abstract:

Brucellosis is a zoonotic disease caused by Brucella, which affects the healthy development of animal husbandry, even threatens the health and safety of human beings, and causes serious economic and social losses. Therefore, the prevention and control of Brucellosis is very necessary. In this study, based on Omp2a, a conserved gene of Brucella, three pairs of primers were designed by online website, and the reaction conditions and reaction system were optimized. At the same time, the established LAMP method was compared with PCR detection method, and was used for the detection of actual samples by Center for Disease Control and Prevention of Shaanxi. The results showed that the amplification results of LAMP could be observed by naked eyes based on HNB indicator. The LAMP detection method can accurately distinguish Brucella from non-Brucella, and the detection limit is 2.56×10-4 ng/μL. In the actual sample detection, the sensitivity and specificity of LAMP were 100% compared with conventional separation and identification methods. Therefore, the LAMP detection method established in this study has the advantages of strong specificity and high sensitivity. The detection results can be observed by naked eye after 45 min amplification, which is suitable for field and grass-roots detection.