食品科学 ›› 2017, Vol. 38 ›› Issue (6): 295-302.doi: 10.7506/spkx1002-6630-201706046

• 安全检测 • 上一篇    下一篇

多重PCR法检测鲜切哈密瓜中3 种食源性致病菌

冯 可,胡文忠,姜爱丽,萨仁高娃,徐永平,杨 柳,王 馨   

  1. 1.大连理工大学生命科学与技术学院,辽宁 大连 116024;2.大连民族大学生命科学学院,辽宁 大连 116600
  • 出版日期:2017-03-25 发布日期:2017-03-28
  • 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)

Establishment of Multiplex PCR Detection Method for Three Foodborne Pathogens on Fresh-Cut Cantaloupe

FENG Ke, HU Wenzhong, JIANG Aili, Sarengaowa, XU Yongping, YANG Liu, WANG Xin     

  1. 1. School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China; 2. College of Life Science, Dalian Minzu University, Dalian 116600, China
  • Online:2017-03-25 Published:2017-03-28

摘要: 建立可同时检测鲜切哈密瓜中的单增李斯特菌、鼠伤寒沙门氏菌和大肠杆菌O157:H7的多重聚合酶链式反应(polymerase chain reaction,PCR)检测方法。根据单增李斯特菌inlA基因、鼠伤寒沙门氏菌invA基因、大肠杆菌O157:H7的wzy基因设计3 对特异性引物。对多重PCR反应体系中的引物浓度、退火温度、Mg2+浓度、dNTP浓度进行了优化,并确定适宜的多重PCR反应体系及反应条件。结果表明:25 μL反应体系,10×PCR buffer为2.5 μL,MgCl2(25 mmol/L)为3.5 μL,dNTPs(2.5 mmol/L)为2 μL,inlA基因上下游引物(5 μmol/L)为1 μL,invA基因上下游引物(5 μmol/L)为1 μL,wzy基因上下游引物(5 μmol/L)为2 μL,单增李斯特菌DNA模板为1 μL,鼠伤寒沙门氏菌DNA模板为1 μL,大肠杆菌O157:H7 DNA模板为1 μL,exTaq DNA聚合酶(5 U/L)为0.3 μL,加ddH2O补足25 μL。反应条件为95 ℃预变性3 min;94 ℃变性30 s,53.9 ℃退火30 s,72 ℃延伸30 s,32 个循环;72 ℃延伸10 min。鲜切哈密瓜中人工接种的目标菌的灵敏度为单增李斯特菌2.7×104 CFU/g,大肠杆菌O157:H7 3.3×104 CFU/g以及鼠伤寒沙门氏菌3.8×104 CFU/g。该技术可为快速检测鲜切哈密瓜中病原菌污染度及其控制提供参考依据。

关键词: 多重PCR, 快速检测, 食源性致病菌, 鲜切哈密瓜

Abstract: The objective of this study was to establish a multiplex polymerase chain reaction (PCR) detection method for Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 on fresh-cut cantaloupe. Three pairs of specific primers were designed according to the inlA gene of L. monocytogenes, the invA gene of Salmonella typhimurium, and the wzy gene of E. coli O157:H7. The primer concentration, annealing temperature, Mg2+ concentration and dNTP concentration were optimized. The results showed that the PCR reactions were performed in a total volume of 25 μL consisting of 2.5 μL of 10 × PCR buffer, 3.5 μL of 25 mmol/L MgCl2, 2 μL of 2.5 mmol/L dNTPs, 1 μL of DNA template, 0.3 μL of exTaq DNA polymerase, 1 μL of 5 μmol/L upstream and downstream primers for inlA and invA and 2 μL of 5 μmol/L upstream and downstream primers for wzy made up to 25 μL with ddH2O. The reaction conditions were as follows: pre-degeneration at 95 ℃ for 3 min followed by 32 cycles of degeneration at 94 ℃ for 30 s, annealing at 53.9 ℃ for 30 s and extension at 72 ℃ for 30 s and a final extension step at 72 ℃ for 10 min. The sensitively of the multiplex PCR was 2.7 × 104 CFU/g for L. monocytogenes, 3.3 × 104 CFU/g (E. coli O157:H7) and 3.8 × 104 CFU/g Salmonella typhimurium inoculated onto freshcut cantaloupe. This method can lay the basis to detect and control these pathogenic microorganisms on fresh-cut cantaloupe.

Key words: multiplex PCR, rapid detection, foodborne pathogen, fresh-cut cantaloupe

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