鲜切果蔬中4种病原微生物多重PCR检测技术

摘要

The objective of this study is to establish a multiplex PCR assay for the detection Listeria monocytogenes,Salmonella typhimurium,Staphylococcus aureus and Escherichia coli O157∶H7 on fresh-cut fruits and vegetables.Four pairs of specific primers were designed according to the inlA gene ofL.monocytogenes,the invA gene of S.typhimurium,the nuc of S.aureus and the wzy gene ofE.coli O157∶H7,respectively.The limits of detection (LODs) for L.monocytogenes,S.typhimurium,S.aureus and E.coli O157∶H7 were 3.5 × 106,1.6 × 10s,2.4 × 105,and 4.8 × 105 CFU/mL,respectively.This assay was advantageous for saving labor,reagents and time (9-11 h vs.5-7 d) over traditional culture method.The present method can provide a significant guidance for enterprises or analytical and testing centers to monitor massive samples.%研发可同时检测鲜切果蔬中的单核细胞性李斯特菌、鼠伤寒沙门菌、金黄色葡萄球菌和大肠杆菌O157∶H7的多重聚合酶链式反应(polymerase chain reaction,PCR)检测方法.根据单核细胞性李斯特菌加inlA基因、鼠伤寒沙门菌invA基因、大肠杆菌O157∶H7 wzy基因、金黄色葡萄球菌nuc基因设计及筛选出4对引物.对多重PCR体系及条件进行优化.该方法对单核细胞性李斯特菌、鼠伤寒沙门菌、金黄色葡萄球菌和大肠杆菌O157∶H7的检出限分别为3.5×106、1.6×105、2.4×105、4.8×105 CFU/mL.将优化的多重PCR方法对不同接种量富集后验证,结果表明,经过9h富集后,该方法检出限为1CFU/mL.该方法在鲜切莴苣、鲜切黄瓜、鲜切木瓜、鲜切哈密瓜中应用同样可扩增出4条目标菌.因此,利用所建立的多重PCR方法对鲜切果蔬中侵染的病原菌检出限可达到1 CFU/g.该方法相较于传统的培养检测方法具有节约大量的劳力、试剂、时间等优点,检测时间也由原来的5～7 d缩短至9～11h,对于企业或分析检验中心大批量样品的监测具有指导意义.