Use of a novel metal indicator to judge loop-mediated isothermal amplification for detecting the 35S promoter
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- 作者：Xiaofu Wang ; Zhenfang Fu ; Xiaoyun Chen…
- 关键词：Loop ; mediated isothermal amplification (LAMP) ; Acid Chrome Blue K (ACBK) ; Metal indicator ; Colorimetric ; 35S promoter
- 刊名：Analytical and Bioanalytical Chemistry
- 出版年：2017
- 出版时间：February 2017
- 年：2017
- 卷：409
- 期：4
- 页码：881-889
- 全文大小：
- 刊物类别：Chemistry and Materials Science
- 刊物主题：Analytical Chemistry; Biochemistry, general; Laboratory Medicine; Characterization and Evaluation of Materials; Food Science; Monitoring/Environmental Analysis;
- 出版者：Springer Berlin Heidelberg
- ISSN：1618-2650
- 卷排序：409
文摘
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.