Department of Chemistry, University of Virginia, McCormick Road, P.O. Box 400319, Charlottesville, Virginia 22904, United States.
New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, United States.
Anal Chem. 2020 Oct 6;92(19):13343-13353. doi: 10.1021/acs.analchem.0c02666. Epub 2020 Sep 21.
Loop-mediated isothermal amplification (LAMP) as a diagnostic tool is rapidly gaining recognition and maturity. Among various advantages over traditional polymerase chain reaction, the ability to visually detect amplification by the incorporation of colorimetric indicators is one of its most unique features. There is an overwhelming variety of LAMP indicators in the literature, yet a comprehensive comparative study is lacking. This study evaluates the use of hydroxynaphthol blue, phenol red, calcein, leuco crystal violet, malachite green, and a fluorescent dye for visual detection. A method for objective quantitative analysis using ImageJ is described that is readily implemented in standard and microfluidic workflows. The work here also includes the largest inter-reader variability study involving 24 participants to evaluate these indicators. We found inaccuracies in visual assessment as bias and/or individual-based perception can exist, solidifying the need for objective analysis. There was not a "universal" indicator, although considerations in sample preparation, storage, and applicability are discussed in length.
环介导等温扩增 (LAMP) 作为一种诊断工具正在迅速得到认可和成熟。与传统聚合酶链反应相比，其具有多种优势，其中通过显色指示剂的掺入来进行可视化扩增检测是其最独特的特征之一。文献中存在大量的 LAMP 指示剂，但缺乏全面的比较研究。本研究评估了使用羟基萘蓝、苯酚红、钙黄绿素、隐色结晶紫、孔雀石绿和荧光染料进行可视化检测。描述了一种使用 ImageJ 进行客观定量分析的方法，该方法易于在标准和微流控工作流程中实施。这里的工作还包括涉及 24 名参与者的最大的读者间变异性研究，以评估这些指示剂。我们发现视觉评估存在不准确之处，因为可能存在偏差和/或基于个体的感知，这就需要进行客观分析。没有“通用”指示剂，尽管在很长篇幅中讨论了样品制备、储存和适用性方面的考虑因素。
