多重实时荧光定量PCR同时检测冷冻蔬菜中4种食源性致病菌

Simultaneous determination of 4 kinds of foodborne pathogens in frozen vegetables by multiplex quantitative real-time PCR

目的建立一种基于TaqMan探针的多重实时荧光定量PCR(multiplex quantitative real-timePCR,multiplex qPCR)同时检测冷冻蔬菜中4种食源性致病菌的方法。方法针对金黄色葡萄球菌nuc基因、痢疾志贺菌rfc基因、沙门氏菌invA基因、单增李斯特氏菌hly基因,设计4对引物和探针,优化反应体系,建立稳定的多重qPCR反应体系。通过阳性菌株污染的方法验证体系的特异性,并确定了冷冻蔬菜样品在细菌水平的检出限。结果各对引物和探针对目标菌有较强的特异性,对其他非目标菌进行检测均未检出,人工污染冷冻蔬菜中志贺菌的检出限为10~2CFU/g,沙门氏菌和单增李斯特氏菌的检出限均为10~3 CFU/g,金黄色葡萄球菌的检出限为10~4 CFU/g。结论本研究可以实现冷冻蔬菜样品中4种致病菌qPCR高效检测。

Objective To establish a method of simultaneous determination of 4 kinds of foodborne pathogens in frozen vegetables by multiplex quantitative real-time PCR by based on TaqMan probes multiplex qPCR. Methods Four pairs of primers and probes were designed for the Staphylococcus aureus nuc gene, Shigella dysenteriae rfc gene, Salmonella invA gene, and Listeria monocytogenes hly gene, and the reaction system was optimized to establish a stable multiple qPCR reaction system. The specificity of the system was verified by the method of contamination by positive strains, and the limit of detection of frozen vegetable samples at the bacterial level was determined. Results Each pair of primers and probes had strong specificity to the target bacteria, and no other non-target bacteria had been detected. The limit of detection of Shigella in artificially contaminated frozen vegetables was 102 CFU/g, the limit of detection of Salmonella and Listeria monocytogenes was 103 CFU/g, and the limit of detection of Staphylococcus aureus was 104 CFU/g. Conclusion This study can achieve high-efficiency detection of 4 kinds of pathogenic bacteria qPCR in frozen vegetable samples.