牛乳中蜡样芽孢杆菌高效环介导扩增检测方法建立

Establishment of efficient loop mediated amplification method for Bacillus cereus detection in milk

中文关键词:  环介导恒温扩增  蜡样芽孢杆菌  牛乳  灵敏度

英文关键词:loop mediated isothermal amplification  Bacillus cereus  milk  sensitivity

中文摘要:

      目的 建立稳定的环介导恒温扩增体系(loop mediated isothermal amplification, LAMP)检测乳制品中蜡样芽孢杆菌的分析方法。方法 设计5组引物, 通过特异性、灵敏度等指标测试筛选出较好的引物对。后通过优化反应中关键试剂及浓度, 检出蜡样芽孢杆菌。通过基因组干扰实验排除引物的假阳性干扰。并在牛奶中验证该方法的检测效率。结果 最终筛选出一组特异性好的引物, 该扩增方法经过优化后, 在最适扩增体系可以检出10 fg/μL纯基因组DNA, 用实时荧光定量实验进行对比验证可以检测出100 ag/μL的基因组DNA。抗干扰实验结果显示干扰组基因组核酸和培养菌液均对标准组无影响。该方法在检测牛乳中混合的菌落时呈现出良好的检测效果, 可以检测出2.1×102 CFU/mL。结论 本研究建立了高效、稳定、灵敏的恒温扩增体系, 可以应用与牛奶中中蜡样芽孢杆菌的快速检测。

英文摘要:

      Objective To establish a stable method for the detection of Bacillus cereus in dairy products by loop mediated isothermal amplification (LAMP) system. Methods Five groups of primers were designed, and better primer pairs were screened out through specificity, sensitivity and other index tests. After that, Bacillus cereus was detected by optimizing the key reagents and concentrations in the reaction. The false positive interference of primers was eliminated through genome interference experiment. The detection efficiency of the method was verified in milk. Results A group of primers with good specificity were screened out. After optimization, the amplification method could detect 10 fg/μL pure genomic DNA in the optimal amplification system, and 100 ag/μL genomic DNA could be detected by real-time fluorescence quantitative experiments. The results of anti-interference experiment showed that the genomic nucleic acid and culture liquid of interference group had no effect on the standard group. The method showed good detection effect when detecting mixed colonies in milk, and could detect 2.1×102 CFU/mL. Conclusion This study establish an efficient, stable and sensitive constant temperature amplification system, which can be applied to the rapid detection of Bacillus cereus in milk.