基于布鲁菌bp26基因LAMP检测方法的建立及优化

Establishment and optimization of LAMP detection system in Brucella based on bp26 gene

布鲁菌病严重影响畜牧业发展和公共卫生安全,及时、准确地检测布鲁菌是该病防控的关键。本研究基于布鲁菌bp26基因设计特异性的环介导等温扩增(LAMP)引物,建立布鲁菌LAMP检测方法。为获得最佳反应条件,对镁离子浓度、LAMP引物比例、反应设备等条件进行优化,对建立的LAMP检测方法的灵敏度、特异性进行评价。结果显示,建立的LAMP反应体系中镁离子浓度为8 mmol/L,内外引物比为6∶1时为最佳体系;分别在金属浴、恒温水浴锅、烘箱等不同环境下实验,结果无差异;对大肠杆菌、肺炎链球菌、金黄色葡萄球菌等8种不同细菌的特异性检测结果显示,该方法特异性良好。在最佳反应条件下,检测灵敏度为10 pg/μL。为获得良好的可视化检测效果,通过对反应体系中不同浓度的锰离子浓度进行优化,结果显示,锰离子浓度为0.1 mmol/L时可以建立最佳可视化效果。本研究建立的布鲁菌LAMP检测方法特异、灵敏、可视化,为布鲁菌的临床快速检测提供了技术支持。

Brucellosis seriously affects the development of animal husbandry and public health safety.Timely and accurate detection of brucellosis is the key to the prevention and control of the disease.The LAMP(loop-mediated isothermal amplification)primers were designed based on the Brucella bp26 gene,to establish and optimize the Brucella LAMP assay in this study.To obtain the best reaction conditions,magnesium ion concentration,LAMP primer concentration,reaction equipment,and other reaction conditions were optimized,and the sensitivity and specificity of the LAMP assay were evaluated,respectively.The optimal LAMP assay was established when the magnesium ion concentration was 8 mmol/L and the ratio of internal and external primers was 6∶1.There was no difference in the metal bath,thermostatic water bath,and oven.This method could only detect Brucella and does not react with Escherichia coli,Streptococcus pneumoniae,and Staphylococcus aureus etc,indicating that the established LAMP assay had good specificity.Under the optimal reaction conditions,the detection sensitivity was 10 pg/μL.For obtaining a good visual detection effect,the concentration of different concentrations of manganese ions was optimized in the reaction system,and the results showed that 0.1 mmol/L manganese ions could establish the best visual effect.The Brucella LAMP assay is specific,sensitive,and visual,providing technical support for rapid clinical detection of Brucella.