关键词: 多响应曲面优化, 毛细管电泳, 激光诱导荧光检测, 食源性致病菌, 多重PCR

Abstract:

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.

Key words: Multi-response optimization, Capillary electrophoresis, Laser-induced fluorescence detection, Foodborne pathogenic bacteria, Multiplex polymerase chain reaction