2. Academic Validation
  3. Use of a novel metal indicator to judge loop-mediated isothermal amplification for detecting the 35S promoter

Use of a novel metal indicator to judge loop-mediated isothermal amplification for detecting the 35S promoter

  • Anal Bioanal Chem. 2017 Feb;409(4):881-889. doi: 10.1007/s00216-016-0084-x.
Xiaofu Wang 1 2,  Zhenfang Fu 3,  Xiaoyun Chen 1 2,  Cheng Peng 1 2,  Xiaoli Xu 1 2,  Wei Wei 1 2,  Feiwu Li 4,  Junfeng Xu 5 6
Affiliations

Affiliations

  • 1 Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang, 310021, China.
  • 2 State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Hangzhou, Zhejiang, 310021, China.
  • 3 College of Chemistry and Life Science, Shenyang Normal University, Shenyang, Liaoning, 110034, China.
  • 4 Institute of Agricultural Standard and Testing Technology, Jilin Academy of Agricultural Sciences, Changchun, Jilin, 130033, China.
  • 5 Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang, 310021, China. njjfxu@163.com.
  • 6 State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Hangzhou, Zhejiang, 310021, China. njjfxu@163.com.
PMID: 27873004 DOI: 10.1007/s00216-016-0084-x
Abstract

Loop-mediated isothermal amplification (LAMP) is a widely used isothermal nucleic acid amplification method. Here we developed a new closed-tube colorimetric method for judging LAMP with a novel metal indicator. First, the metal indicator, acid chrome blue K (ACBK), was evaluated in the LAMP reaction with various combinations of reaction reagents, such as reaction buffer, dNTP mixtures, primer mixtures, or Mg2+. We found that the solution color of the LAMP reaction with ACBK changed from red to blue based on a decrease in the Mg2+ concentration in the reaction solution. We then optimized the LAMP with ACBK method for detecting the Cauliflower Mosaic Virus 35S promoter. Further, the specificity of the new colorimetric assay using ACBK in the LAMP reaction for detecting the 35S promoter was tested with diverse transgenic events in different crops, and the sensitivity threshold of the assay was ∼50 copies for transgenic rice genomic DNA and 100 ng of 0.1 % DNA from rice, soybean, rapeseed, and maize. Finally, the applicability of the LAMP assay was successfully validated using practical maize samples. All the detection results could be easily discerned either by UV-vis spectroscopy or the naked eye. Graphical Abstract The visual detect LAMP amplification by the addition of ACBK as a signal indicator. The color of the LAMP-ACBK solution turned from red to blue as the concentration of free Mg2+ decreases. The detection results could be easily discerned either by UV-vis spectroscopy or the naked eye.

Keywords

35S promoter; Acid Chrome Blue K (ACBK); Colorimetric; Loop-mediated isothermal amplification (LAMP); Metal indicator.

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