Establishment and optimization of LAMP detection system in Brucella based on bp26 gene
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 reac-tion 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,ther-mostatic water bath,and oven.This method could only detect Brucella and does not react with Es-cherichia coli,Streptococcus pneumoniae,and Staphylococcus aureus etc,indicating that the estab-lished LAMP assay had good specificity.Under the optimal reaction conditions,the detection sensitivi-ty was 10 pg/μL.For obtaining a good visual detection effect,the concentration of different concen-trations 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.
国家科技期刊平台
NSTL
万方数据
