1. Use of a novel metal indicator to judge loop-mediated isothermal amplification for detecting the 35S promoter
- Author
-
Xu Xiaoli, Wang Xiaofu, Wei Wei, Peng Cheng, Feiwu Li, Chen Xiaoyun, Xu Junfeng, and Zhenfang Fu
- Subjects
0301 basic medicine ,Loop-mediated isothermal amplification ,Analytical chemistry ,Polymerase Chain Reaction ,01 natural sciences ,Biochemistry ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Caulimovirus ,Limit of Detection ,Promoter Regions, Genetic ,Electrophoresis, Agar Gel ,Chromatography ,biology ,010401 analytical chemistry ,Plants, Genetically Modified ,biology.organism_classification ,eye diseases ,0104 chemical sciences ,genomic DNA ,030104 developmental biology ,chemistry ,Metals ,Reagent ,Nucleic acid ,Colorimetry ,Spectrophotometry, Ultraviolet ,sense organs ,Cauliflower mosaic virus ,Naked eye ,Primer (molecular biology) ,DNA - 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.
- Published
- 2016
- Full Text
- View/download PDF