食品科学 ›› 2018, Vol. 39 ›› Issue (6): 276-283.doi: 10.7506/spkx1002-6630-201806043

• 安全检测 • 上一篇    下一篇

鲜切果蔬中4 种病原微生物多重PCR检测技术

冯可1,2,胡文忠1,*,姜爱丽1,萨仁高娃1,2,徐永平2,司琦1,马新秀1   

  1. (1.大连民族大学生命科学学院,生物技术与资源利用教育部重点实验室,辽宁?大连 116600;2.大连理工大学生命科学与技术学院,辽宁?大连 116024)
  • 出版日期:2018-03-25 发布日期:2018-03-14
  • 基金资助:
    “十三五”国家重点研发计划重点专项(2016YFD0400903);国家自然科学基金面上项目(31471923;31172009); “十二五”国家科技支撑计划项目(2012BAD38B05)

Multiplex PCR Method for Detection of Four Foodborne Pathogens on Fresh-Cut Fruits and Vegetables

FENG Ke1,2, HU Wenzhong1,*, JIANG Aili1, Sarengaowa1,2, XU Yongping2, SI Qi1, MA Xinxiu1   

  1. (1. Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, China; 2. School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China)
  • Online:2018-03-25 Published:2018-03-14

摘要: 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9?h富集后,该方法检出限为1?CFU/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9~11?h,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。

关键词: 多重PCR, 快速检测, 食源性病原微生物, 鲜切果蔬

Abstract: 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 of L. monocytogenes, the invA gene of S. typhimurium, the nuc of S. aureus and the wzy gene of E. 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 × 105, 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.

Key words: multiplex PCR, rapid detection, foodborne pathogen, fresh-cut fruits and vegetables

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