氨基化磁珠非靶向富集食源性致病菌的条件筛选及优化

Screening and Optimization of Conditions for Untargeted Enrichment of Foodborne Pathogens by Amino Magnetic Beads

该研究基于氨基化磁珠静电富集细菌技术,建立了一种可同时富集鼠伤寒沙门氏菌,金黄色葡萄球菌,单增李斯特氏菌的方法。在每2 mL单菌液中(103 CFU/mL)分别加入5~200μg的3种粒径的氨基化磁珠(1μm、300 nm、100 nm),当孵育5~90 min时检测各细菌的富集效率。将单菌液体系的富集正交试验结果应用至混和菌体系的单因素试验和正交试验,将最终结果应用至大体积实验体系以及实际样本(市售牛奶、水果沙拉)。结果表明,当氨基化磁珠添加量为50μg、孵育时间为30 min时对3种病原菌可以达到60%以上的捕获率,所有反应均在pH值7.4的PBS中进行。磁珠粒径选择300 nm(p<0.05)。在实际样本牛奶、水果沙拉中,当三种混合菌的最低终浓度为2×102 CFU/g(mL)时,捕获率高于55%,结果可达到荧光定量PCR的检测低限。氨基化磁珠与免疫磁珠比较,其具有稳定保存、低成本,高效率等优势,其非靶向富集的能力为下游食源性致病菌的快速检测提供有效前处理富集手段。

In this study,a method for the simultaneous enrichment of Salmonella typhimurium,Staphylococcus aureus and Listeria monocytogenes was developed based on the electrostatic enrichment technology using amino magnetic beads.Three different sized amino magnetic beads(5~200μg;1μm,300 nm,100 nm)were added respectively into each bacterial solution(2 mL;103 CFU/mL),and the enrichment efficiency of each bacteria was detected after the incubation was conducted for 5~90 min.The results of the orthogonal enrichment tests for the single bacteria system were applied to the single factor tests and orthogonal tests of the mixed bacteria system,and the final results were applied to the large-volume experimental system and actual samples(commercially available milk and fruit salad).The results showed that when the addition amount of amino magnetic beads was 50μg and the incubation time was 30 min,the capture rate for the three kinds of pathogens could reach over 60%.All reactions were performed in PBS at pH 7.4.The bead diameter was selected to be 300 nm(p<0.05).The capture rate was higher than 55%when the minimum concentration of the three mixed bacteria was 2×102 CFU/g(mL)in the actual samples of milk and fruit salad.This result could reach the detection limit of fluorescence quantitative PCR.Compared with immunomagnetic beads,the amino magnetic beads had many advantages,such as high stability over storage,low cost and high efficiency.The ability of untargeted enrichment provides an effective pretreatment enrichment method for the rapid detection of downstream foodborne pathogens.