多重PCR同时检测食品中4种细菌与常见霉菌

Multiplex PCR for Simultaneous Detection of Four Food-Borne Bacteria and Common Mold

建立一种同时检测食品中金黄色葡萄球菌、单核细胞增生李斯特氏菌、沙门氏菌、大肠杆菌O157:H7以及霉菌的多重聚合酶链式反应(polymerase chain reaction,PCR)检测方法。根据金黄色葡萄球菌的耐热核酸酶基因(nuc)、沙门氏菌的侵袭正调节蛋白基因(hilA)、大肠杆菌O157:H7鞭毛基因(flic)、单核细胞增生李斯特氏菌的毒力调控蛋白基因(prfA)及霉菌18S rRNA基因V5区分别设计5对特异性引物,并对PCR扩增反应体系和扩增条件进行优化,确定获得特异性良好的PCR扩增产物。而后在37℃对人工污染致病菌的香肠、面包和豆腐进行增菌培养,5种致病微生物在20 h内的检出限均可达到100 CFU/25 g。本实验建立的多重PCR检测方法适用于食品中4种细菌与常见霉菌的同时检测,相较于传统检测方法,具有快速、简便、特异性高的优点。

In this study,a multiplex polymerase chain reaction(PCR)method was developed to simultaneously detect Staphylococcus aureus,Listeria monocytogenes,Salmonella,Escherichia coli O157:H7,and mold in foods.Speciesspecific primers were designed targeting the heat-resistant nuclease gene(nuc)in S.aureus,the invader upregulation protein gene(hilA)in Salmonella,the flagellum gene(flic)in E.coli O157:H7,the virulence regulation protein gene(prfA)in L.monocytogenes and the V5 region of the 18S rRNA gene in mold,and the PCR reaction system and conditions were optimized for highly specific amplification.Sausage,bread and tofu artificially contaminated with the five foodborne pathogens were enriched at 37℃.The detection limits of the five food-borne pathogens within 20 hours all reached 10^0 CFU/25 g.The multiplex PCR method established in this study is suitable for rapid detection of common pathogenic organisms in foods.Compared with traditional detection methods,it has the advantages of rapidity,simplicity and high specificity and thus,can be used to simultaneously detect bacteria and mold in foods.