食品科学

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婴儿配方乳粉中阪崎克罗诺杆菌解旋酶恒温基因扩增检测方法的建立

周 巍1,2,张 薇2,刘 亮2,刘 东2,李永波2,田 浩2,张 岩2,*,张志胜1,*   

  1. 1.河北农业大学食品科技学院,河北 保定 071000;2.河北省食品质量监督检验研究院,河北 石家庄 050091
  • 出版日期:2014-02-25 发布日期:2014-03-17
  • 通讯作者: 张 岩
  • 基金资助:

    河北省质量技术监督局科技计划项目(100108)

Detection of Cronobacter sakazakii in Infant Formula Powder by Helicase-Dependent Isothermal DNA Amplification Assay

ZHOU Wei1,2, ZHANG Wei2, LIU Liang2, LIU Dong2, LI Yong-bo2, TIAN Hao2, ZHANG Yan2,*, ZHANG Zhi-sheng1,*   

  1. 1. College of Food Science and Technology, Agricultural University of Hebei, Baoding 071000, China;
    2. Hebei Institute of Food Quality Supervision Inspection and Research, Shijiazhuang 050091, China
  • Online:2014-02-25 Published:2014-03-17
  • Contact: ZHANG Yan

摘要:

目的:建立一种检测婴儿配方乳粉中阪崎克罗诺杆菌的解旋酶恒温基因扩增方法。方法:根据阪崎克罗诺杆菌ITS基因设计特异性引物,优化解旋酶恒温基因扩增法反应条件UvrD helicase、T4 gp32的浓度,人工添加阪崎克罗诺杆菌确定检出限,多种致病菌在建立的解旋酶恒温基因扩增体系中扩增验证特异性,电泳检测扩增产物。结果:解旋酶恒温基因扩增法检测婴儿配方乳粉中阪崎克罗诺杆菌得到与设计序列长度一致的100 bp基因片段,检出限为10 CFU/g,优化反应条件UvrD helicase、T4 gp32的终浓度分别为0.1、5.0 μg。结论:解旋酶恒温基因扩增法用于检测婴儿配方乳粉中阪崎克罗诺杆菌的特异性强、灵敏度高、耗时短,为婴儿配方乳粉中阪崎克罗诺杆菌的快速检测提供了新的方法。

关键词: 解旋酶恒温基因扩增法, 婴儿配方乳粉, 阪崎克罗诺杆菌, 检测

Abstract:

Objective: The aim of this study was to establish a helicase-dependent isothermal DNA amplification
(HAD) method for rapid and accurate detection of Cronobacter sakazakii in infant formula powder. Methods: A pair of
oligonucleotide primers exclusively targeting the internal transcribed spacer (ITS) gene of Cronobacter sakazakii was
designed, and the final concentration of UvrD helicase and T4 gp32 were optimized. Cronobacter sakazakii in infant formula
powder was then directly detected by this method and amplification products were separated and detected by agarose gel
electrophoresis. Results: An amplicon of 100 bp in length was obtained, which was the same as the designed gene fragment
by HDA. The sensitivity of HDA was 101 CFU/g, the optimized concentration of UvrD helicase was 0.1 μg, and T4 gp32
was 5.0 μg. Conclusion: The helicase-dependent isothermal DNA amplification assay provides a new, specific, sensitive and
fast for the detection of Cronobacter sakazakii in infant formula powder.

Key words: helicase-dependent isothermal DNA amplification (HDA), infant formula powder, Cronobacter sakazakii, detection

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