Division Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Hochschulring 1, 15745 Wildau, Germany.
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia.
Sci Rep. 2017 Mar 24;7:45085. doi: 10.1038/srep45085.
We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.
我们发现茜素红 S 和其他知名荧光染料可用于酶法检测中焦磷酸根的在线检测，包括环介导等温扩增 (LAMP) 和聚合酶链反应 (PCR) 检测。我们采用迭代筛选的方法对一组选定的化合物进行了筛选，首先确保酶的兼容性，评估在锰作为猝灭剂存在的情况下对无机焦磷酸根的敏感性，并优化在线检测的条件。在所选择的染料中，廉价的茜素红 S 被发现可在 LAMP 和 PCR 条件下选择性地检测焦磷酸根，并且在其定义的红移光谱、长保质期和低毒性方面具有优势。此外，新发现的性质也可能对其他不产生核酸但基于无机焦磷酸根的酶法检测有用。最后，我们提出，我们的筛选方法可能为快速筛选用于检测无机焦磷酸根的化合物提供蓝图。
