食源性致病菌多重PCR检测技术建立及其应用
Development and application of multiplex PCR detection techniques for foodborne pathogenic bacteria
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摘要: 针对6种食源性致病菌特异性基因（金黄色葡萄球菌nuc基因、副溶血性弧菌tdh基因、单核细胞增生李斯特菌hly基因、阪崎克罗诺杆菌ompA基因、鼠伤寒沙门氏菌invA基因和大肠杆菌O157：H7 rfbE基因）建立了多重PCR检测技术，分析了其特异性和敏感性，并评估了其在人工染菌牛奶中的应用可行性。结果表明：该检测技术可扩增预期的345 bp、307 bp、269 bp、237 bp、188 bp和142 bp细菌特异性PCR产物，无非特异性扩增，且不与非靶细菌产生交叉反应；对细菌纯培养物基因组DNA的检测限为10 pg，对人工染菌牛奶样品中致病菌的检测限为103 CFU/mL，可实现6种食源性致病菌特异性强、灵敏度高、简便高效的同时检测。Abstract: Multiple PCR detection techniques were established for 6 foodborne pathogenic bacteria specific genes (the nuc gene of Staphylococcus aureus, tdh gene of Vibrio parahaemolyticus, hly gene of Listeria monocytogenes, ompA gene of Cronobacter sakazakii, invA gene of Salmonella typhimurium, and rfbE gene of Escherichia coli O157:H7). The sensitivity and specificity of multiplex PCR were analyzed, and its application feasibility in artificially-contaminated milk was also evaluated. The results showed that the specific PCR products of 345 bp, 307 bp, 269 bp, 237 bp, 188 bp and 142 bp were amplified as expected in multiplex PCR without nonspecific amplification and cross reactivity with non-target pathogenic bacteria. Multiplex PCR developed in this study could detect as little as 10 pg of genomic DNA of pure bacterial culture and 103 CFU/mL of milk artificially-contaminated with pathogenic bacteria, which could specifically, sensitively, rapidly and efficiently detect six foodborne pathogenic bacteria simultaneously.
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Key words:
- foodborne pathogenic bacteria /
- multiplex PCR /
- detection techniques
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