多重PCR快速检测3种食源性致病菌

Detection of three food-borne pathogenic microorganisms by multiplex PCR

目的建立一种能同时检测金黄色葡萄球菌、产单核李斯特菌和沙门菌3种致病菌的多重PCR检测方法。方法采用LB培养液对金黄色葡萄球菌、产单核李斯特菌和沙门菌标准菌株进行增菌。根据金黄色葡萄球菌的nuc基因、产单核李斯特氏菌的hlyA基因、沙门氏菌的invA基因设计引物,通过多重聚合酶链反应(PCR)对上述3种食源性致病菌的目的基因进行扩增,同时对反应体系进行优化。结果对平均浓度为5cfu/ml的金黄色葡萄球菌、产单核李斯特菌和沙门氏菌在LB培养液中进行8h振荡培养,可以检出阳性结果;把金黄色葡萄球菌、产单核李斯特菌、沙门菌、志贺菌、蜡样芽孢杆菌、大肠埃希菌O157、阪崎肠杆菌7种菌混合在一起提取混合基因组DNA进行PCR扩增,显示出很好的特异性结果。结论建立的多重PCR检测方法适用于金黄色葡萄球菌、产单核李斯特菌和沙门菌的快速检测,具有快速、简便、灵敏的特点,可广泛应用于食品卫生检测、食物中毒应急处理和临床检验等领域。

Objective To establish a multiplex PCR method for simultaneous detection of Staphylococcus aures, Listeria monocytogens and Salmonella spp. in food. Methods Staphylococcus aures, Listeria monocytogens, and Salmonella spp. were en- riched by LB medium. The primers were designed according to nuc gene of Staphylococcus aures, hlyA gene of Listeria monocyto- gens and invA gene of Salmonella spp. The target genes of these pathogens in food were amplified by multiplex PCR, whose reac- tion conditions were optimized specifically. Results The multiplex PCR method established in this experiment had high specificity while seven kinds of microorganism DNA were mixed in one PCR reaction tube, and the detection limit of the method was 5 cfu/ml for Staphylococcus aures, Listeria monocytogens and Salmonella spp. Conclusion The multiplex PCR method, which was rapid, convenient and had high sensitivity, could be used in fields such as food sanitation detection, foodborne illness detection and clini- cal inspection.