多响应曲面优化-毛细管电泳-激光诱导荧光快速检测食源性致病菌

建立了食品中常见致病菌: 沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR产物-毛细管电泳快速检测方法. 根据沙门菌、大肠杆菌O157∶H7、志贺菌及副溶血性弧菌的特异性基因保守序列设计出多重PCR引物, 优化PCR 扩增反应体系. 采用多响应曲面法优化毛细管电泳的分离条件, 以含有DNA荧光染料SYBR Green Ⅰ的1.0％甲基纤维素为筛分介质, 通过毛细管电泳-激光诱导荧光同时检测4种常见致病菌的PCR 扩增产物. 在优化的多重PCR反应体系和毛细管筛分电泳条件下, 此方法可以同时检测出沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR扩增产物, 25 min内即可完成检测. 迁移时间的日内相对标准偏差为0.92%~1.58%. 通过多响应曲面的优化, 有效改善了毛细管电泳对DNA分子的分离能力.

Simultaneous Multi-response Optimization and Capillary Electrophoresis with Laser Induced Fluorescence Detector for Rapid Detection of Foodborne Pathogenic Bacteria

A rapid, sensitive and reliable method for monitoring foodborne pathogenic bacteria by multiplex PCR-capillary electrophoresis with laser induced fluorescence detector was developed. Four sets of primers were designed to simultaneously amplify the gene segments of invA gene in salmonella, rfbO157 gene in E.coli.O157∶H7, ipaH gene in Shigella and Vpara(16S-23S rDNA IGS) gene in Vibrio parahemolyticus and the multiplex PCR system was optimized. The separation conditions of capillary electrophoresis were optimized by central composite design(CCD) and simultaneous multiresponse optimization. The method was applied to the rapid detection of above PCR products by a capillary coated with linear polyacrylamide and 1.0％MC-4000 sieving buffer with SYBR Green Ⅰ as DNA fluorescent dyes under a separation voltage of 5.2 kV. The proposed method was able to simultaneously detect the PCR products of the above four genes under the optimization conditions of PCR reaction and capillary electrophoresis within 25 min. The within-day precisions of migration time were 0.92%-1.58%. Compared with agarose gels electrophresis, the proposed method is rapid, sensitive and accurate.