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色谱

• 研究论文 • 上一篇    下一篇

多重聚合酶链反应-毛细管电泳-激光诱导荧光法检测三种食源性致病菌

毛红霞,黎源倩,裴晓方,何超,渠凌丽   

  1. West China School of Public Health, Sichuan University, Chengdu 610041, China
  • 收稿日期:2007-01-10 修回日期:2007-02-12 出版日期:2007-07-30 发布日期:1985-06-25
  • 通讯作者: 黎源倩

Rapid Detection of Three Foodborne Pathogenic Bacteria by Multiplex Polymerase Chain Reaction-Capillary Electrophoresis with Laser Induced Fluorescence Detector

MAO Hongxia, LI Yuanqian, PEI Xiaofang, HE Chao, QU Lingli   

  1. West China School of Public Health, Sichuan University, Chengdu 610041, China
  • Received:2007-01-10 Revised:2007-02-12 Online:2007-07-30 Published:1985-06-25

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摘要： 建立了食品中常见致病菌大肠杆菌O157:H7的uidA基因、沙门菌的invA基因和志贺菌的ipaH基因的多重聚合酶链反应（PCR）产物的毛细管电泳快速检测方法。根据这3种致病菌的特异性基因序列设计多重PCR引物,优化PCR扩增反应体系,采用7.0 g/L 甲基纤维素为筛分介质,毛细管电泳-激光诱导荧光检测法同时检测了3种常见致病菌的PCR扩增产物。在优化的多重PCR反应和毛细管筛分电泳条件下,该方法可以同时检测沙门菌、志贺菌和大肠杆菌O157:H7基因的多重PCR扩增产物,22 min内即可完成3种常见致病菌的毛细管电泳检测。迁移时间的相对标准偏差为1.47%~2.07%。与凝胶电泳法比较,该法简便快速,灵敏度高,可用于多种致病菌脱氧核糖核酸的检测,为食品安全提供了一种可靠的快速检测方法。

关键词: 多重聚合酶链反应, 激光诱导荧光检测, 毛细管电泳法, 食源性致病菌

Abstract: A method for monitoring foodborne pathogenic bacteria by multiplex polymerase chain reaction （PCR）-capillary electrophoresis (CE) with a laser induced fluorescence detector was developed. Three sets of primers were designed to amplify the gene segments of uidA gene in E.coli.O157:H7, invA gene in salmonella and ipaH gene in Shigella, individually. The multiple PCR system and the separation conditions of CE were optimized. Using a capillary coated with linear polyacrylamide and sieving buffer of 7.0 g/L methyl cellulose (MC) under 8.3 kV of electric voltage, the proposed method was able to simultaneously detect the PCR products of specific genes existing in the three kinds of pathogenic bacteria within 22 min. The relative standard deviations of migration time for the detected DNA fragments were ranging from 1.47% to 2.07%. In comparison with agarose gel electrophoresis, the proposed method is rapid, sensitive and accurate.

Key words: capillary electrophoresis (CE), laser-induced fluorescence detection； foodborne pathogenic bacteria , multiplex polymerase chain reaction (multiplex PCR)