微流控芯片-激光诱导荧光快速检测4种食源性致病菌

Microfluidic Chip Electrophoresis with Laser-Induced Fluorescence Detection for Rapid Analysis of Four Foodborne Pathogenic Bacteria

建立了食品中4种常见食源性致病菌的微流控芯片快速检测方法。根据副溶血弧菌的Vpara（16S-23SrDNAIGS）基因、沙门菌的invA基因、大肠杆菌O157：H7的rfbO157基因和志贺菌的ipaH基因序列设计了4对特异性引物，对上述致病菌进行四重PCR扩增，采用微流控芯片-激光诱导荧光检测食品中4种常见致病菌的多重PCR扩增产物。优化了多重PCR扩增和微流控芯片电泳分离的实验条件。当芯片电泳的筛分介质HPMC-50浓度为2．2％、溴乙锭（EB）含量为3．75μmol／L、电场强度为120V／cm时，pUC Mix DNA Marker8和待测致病菌的多重PCR扩增产物可以实现基线分离，600S内即可完成上述4种致病菌的同时检测，迁移时间的日内相对标准偏差为0．74％～2．09％。本方法能够检出1×10^2cfu／mL的副溶血弧菌、沙门菌、大肠杆菌O157：H7和志贺菌。方法特异性高，所设计的引物在10种非目的菌株体系中均未见扩增的片段。将本法应用于食品中上述致病菌的测定，获得了满意的结果，为常见食源性致病菌的快速检测提供了一种新的可靠分析手段，对保障食品安全具有重要的现实意义。

A rapid and simple method based on a microfluidic chip system with laser-induced fluorescence detection was developed and applied to rapid analysis of qnadruplex polymerase chain reaction （PCR） products for determination of four foodborne pathogenic bacteria. The four specific primer pairs were designed to amplify the target gene fragments： Vpara（16S-23S rDNA IGS）gene of Vibrio parahemolyticus, invA gene of Salmonella, rfbO157 gene of Eseherichia coli O157 ： H7 and ipaH gene of Shigella. Then the quadruplex PCR system was optimized. The multiplex PCR products were separated and detected by mierofluidic chip electrophoresis. Excellent separation was achieved using 2.2% hydroxypropyl methyleellulose-50 （HPMC-50） and 3.75 μmol/L ethidium bromide（EB） at 120 V/cm. The proposed method was employed to detect the quadruplex PCR products of four foodborne pathogenic bacteria simultaneously under the optimal conditions within 600 s. 1 × 10^2 cfu/mL for all four pathogens could he detected. The relative standard deviations of migration time were from 0.7% to 2.1%. No amplification products were obtained using DNA templates from 10 strains of non-target bacteria in the quadruplex PCR reaction, which showed that this method was specific. The present method has been successfully applied to rapid analysis of multiplex PCR products of pathogenic bacteria in artificially inoculated food samples. This study offered another reliable and valuable tool for rapid analysis of foodborne pathogenic bacteria, which was very important for ensuring food safety and security.