水传病原微生物PCR-纳米金显色快速检测方法

Rapid detection of waterborne pathogens using PCR amplification and nanogold visualization

水传病原微生物是腹泻等肠胃疾病发病的主要原因,而发展快速、有效的检测方法对于控制和减少该类微生物的传播风险十分重要.因此,本文研究了PCR反应结合不修饰的纳米金颗粒(Gold nano-particles,AuNPs)显色检测方法的最佳盐浓度、探针含量、PCR反应条件等,并从纳米金制备、病原菌种类、检测特异性等方面进行考核,确定了最优检测条件为:10μL PCR扩增产物混合20 pmol探针,95℃反应5 min,55.5℃反应3 min后,40℃杂交10 min;此时,先后加入90μL实验用纳米金和一定浓度的PBS溶液,室温观察颜色变化.结果发现,纳米金溶液由红色变为紫色或近于无色为阳性结果,对应的吸光度值迁移出530 nm,以此可达到快速检测病原微生物的目的.

Waterborne pathogens are the main cause for diarrhea and other gastrointestinal diseases. It is important to develop a rapid and efficient detection method for controlling them and reducing the risk of their spreading. In this study,the key factors affecting the performance of PCR amplification and nanogold visualization were investigated including the ion concentration,probe concentration and PCR reaction conditions. Furthermore,the preparation of nanogold,the species of the pathogens and the detection specificity were also considered. The optimized protocol was finally established as the following: 10 μL PCR amplicon solution were mixed with 20 pmol probe solution at 95 ℃ for 5 min; 55 ℃,3 min annealing was followed; then the mixture was set at 40 ℃ for 10 min for hybridization; 90 μL nanogold solution and a certain concentration PBS buffer were added finally to observe the color change. A positive result could be confirmed when the color of nanogold solution turned to purple or colorless from red and the OD530 dropped dramatically.