正交优化多重PCR反应体系检测3种食源性致病菌的研究

Application of Orthogonal Design to Optimize Multiplex PCR Reaction System for Detection of Three Food-borne Bacterial Pathogens

[目的]建立金黄色葡萄球菌、沙门氏菌、志贺氏菌的快速、敏感、特异的多重PCR检测方法。[方法]根据金黄色葡萄球菌的nuc基因、沙门氏菌的IpaB基因、志贺氏菌的ipaH基因,设计3对特异性引物进行多重PCR检测,利用正交设计对多重PCR反应体系的镁离子、Taq酶、dNTP、引物的浓度在4个水平上进行L16(44)优化试验,并确定该检测方法的特异性与灵敏度。[结果]3对引物能特异性扩增出210、280、393 bp的目的条带。利用FTA滤膜提取模板DNA,采用建立多重PCR同时对3种食源性致病菌进行检测具有较高的灵敏度,灵敏度金黄色葡萄球菌为3.5×102cfu/ml、沙门氏菌为2.2×102cfu/ml、志贺氏菌为1.0×102cfu/ml。[结论]该检测方法准确、快速、高效,为同时检测食品中多种致病菌奠定了基础。

[ Objective ] The research aimed to establish a rapid, sensitive and specific multiplex PCR method for the simultaneous detection of Staphylococcus aureus, Sahrtonella spp., and Shigella spp. [ Method] Three pairs of specific primers were designed according to Staphylococcus aureus nuc gene, almonella spp. IpaB gene, higella spp. ipaH gene. Orthogonal experimental design was used to optimize multiple PCR amplification system for food - borne bacterial pathogens of four factors （Tatl DNA polymerase, Mg^2＋ , dNTP and primers） at four levels. The specificity and the sensitivity of this detection method were confirmed. [ Result ] Three DNA fragments of 210,280 and 393 bp were amplified. Template was prepared using FTA filter and three food-borne bacterial pathogens were simultaneously detected by the established multiplex PCR technology. The sensitivity of this method was 3.5 × 10^2 cfu/ml for Staphylococcus aureus, 2.2 × 10^2 cfu/ml for Salmonella spp. , and 1.0 × 10^2 cfu/ml for Shigella spp.. [ Conclusion ] This method was accurate, rapid and efficient in the diagnosis, so it could be a useful method for the simultaneous detection of the three species of bacteria in food.