Research and Development of Multiplex Real-time Fluorescence PCR Detection Method for Live Pathogenic Bacteria in Food and Beverage

doi:10.19586/j.2095-2341.2020.0107

Curr. Biotech. ›› 2020, Vol. 10 ›› Issue (6): 717-727.DOI: 10.19586/j.2095-2341.2020.0107

Research and Development of Multiplex Real-time Fluorescence PCR Detection Method for Live Pathogenic Bacteria in Food and Beverage

KE Zhenhua

National Centre for Quality Supervision and Testing of Processed Food (Fuzhou), Fujian Inspection and Research Institute for Product Quality, Fuzhou 350000, China

Received:2020-09-02 Online:2020-11-25 Published:2020-10-01

餐饮及相关食品中病原菌活菌多重实时荧光PCR检测方法的研究与开发

柯振华

福建省产品质量检验研究院，国家加工食品质量监督检验中心(福州)，福州 350000

通讯作者: 柯振华 E-mail: kzhunter@163.com
基金资助:
福建省科技计划项目(2018Y0019)。

Abstract

Abstract: In order to meet the needs of rapid detection of pathogens such as food and beverage, and to establish a pathogen screening method, we selected Shigella dysenteriae, Staphylococcus aureus, Vibrio parahaemolyticus, Enterobacter cloacae, Enterobacter aerogenes, Salmonella, Bacillus cereus, Escherichia coli O157∶H7 and Listeria monocytogenes to carry out multiplex real-time fluorescence PCR methods. To save time and improve efficiency, we developed a general medium for several kinds of bacteria and extracted nucleic acid by high temperature pyrolysis. Screening live bacteria DNA and inactivated dead bacteria by PMA dyes, and detected target bacteria by multiplex real-time fluorescence PCR technology. The detection of target bacteria can be completed within 16 h. The detection limit of target pathogen can reach 103 CFU mL-1.

Key words: food and beverage, PMA-qPCR, pathogenic bacteria, Shigella dysenteriae, Staphylococcus aureus, Vibrio parahaemolyticus, Enterobacter cloacae, Enterobacter aerogenes, Salmonella, Bacillus cereus, Escherichia coli O157∶H7, Listeria monocytogenes

摘要： 为了应对餐饮等食品中病原菌快速检测的需求、研究建立病原菌筛查方法，选取痢疾志贺氏菌（Shigella dysenteriae）、金黄色葡萄球菌（Staphylococcus aureus）、副溶血性弧菌（Vibrio parahaemolyticus）、阴沟肠杆菌（Enterobacter cloacae）、产气肠杆菌（Enterobacter aerogenes）、沙门氏菌（Salmonella）、蜡样芽胞杆菌（Bacillus cereus）、大肠埃希氏菌O157∶H7（Escherichia coli O157∶H7）、单核细胞增生李斯特氏菌（Listeria monocytogenes）等9种病原菌开展多重实时荧光PCR方法研究工作。为了节约预增菌时间与提升检测效率，研发了适用于多种病原菌预增菌的通用型培养基，采取高温裂解法提取菌液核酸，利用PMA染料灭活死亡细菌DNA，筛选出活菌DNA，采用多重实时荧光PCR技术检测目标菌，该方法可在16 h内完成检测，对于目标病原菌的检测低限可达103 CFU·mL-1。

关键词: 餐饮食品, PMA-qPCR, 病原菌, 痢疾志贺氏菌, 金黄色葡萄球菌, 副溶血性弧菌, 阴沟肠杆菌, 产气肠杆菌, 沙门氏菌, 蜡样芽胞杆菌, 大肠埃希氏菌O157∶H7, 单核细胞增生李斯特氏菌

KE Zhenhua. Research and Development of Multiplex Real-time Fluorescence PCR Detection Method for Live Pathogenic Bacteria in Food and Beverage[J]. Curr. Biotech., 2020, 10(6): 717-727.

柯振华. 餐饮及相关食品中病原菌活菌多重实时荧光PCR检测方法的研究与开发[J]. 生物技术进展, 2020, 10(6): 717-727.

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Research and Development of Multiplex Real-time Fluorescence PCR Detection Method for Live Pathogenic Bacteria in Food and Beverage

餐饮及相关食品中病原菌活菌多重实时荧光PCR检测方法的研究与开发

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