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| Application of Metal Indictor Acid Chrome Blue K in TNOS-LAMP Visual Detection System |
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Abstract Loop mediated isothermal amplification (LAMP) is one of isothermal nucleic acid amplification methods that have been widely employed in many areas. In this study, a new closed-tube colorimetric method for monitoring LAMP with a novel metal indicator was developed. Firstly, acid chrome blue K (ACBK), the metal indicator was evaluated in the initial LAMP reaction combined with various combination of reaction ingredient, such as reaction buffer, dNTPs mixture, primers mixture or Mg2+, respectively. This procedure demonstrated that the solution color of the LAMP reaction with ACBK could be changed from red to blue, as the concentration of Mg2+ decreasing in the reaction solution. Subsequently, the concentration of ACBK and Mg2+ in the LAMP detection system was selected and optimized. Finally, selected 2 μL 0.04% ACBK and 6.0 mmol/L Mg2+ in the 25 μL LAMP detection system. The LAMP with ACBK for detection of TNOS was optimized and established. Further, the specificity of the new established colorimetric assay employing ACBK in LAMP reaction for TNOS was tested with diverse transgenic events from different crops, and the sensitivity threshold of the assay was absolute 100 copies for transgenic rice(Oryza sativa) genomic DNA and 50 ng 0.1% DNA from rice, soybean (Glycine max), rape (Brassica napus), and maize(Zea mays), respectively. Finally, the applicability of the LAMP assay was further validated successfully using practical maize samples. All the detection results can be easily discerned either via naked-eye or using UV-vis spectroscopy. The established method can accelerate the speed of detection of genetically modified ingredients as well as provide new idea for judgment of the isothermal nucleic acid amplification.
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Received: 19 January 2016
Published: 22 July 2016
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