Comparative Evaluation and Quantitative Analysis of Loop-Mediated Isothermal Amplification Indicators

Anal Chem. 2020 Oct 6;92(19):13343-13353. doi: 10.1021/acs.analchem.0c02666. Epub 2020 Sep 21.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Colorimetry
  • Fluoresceins / analysis*
  • Fluoresceins / chemistry
  • Fluorescent Dyes / chemistry
  • Gentian Violet / chemistry
  • Humans
  • Indicators and Reagents / chemistry*
  • Lab-On-A-Chip Devices
  • Molecular Diagnostic Techniques*
  • Naphthalenesulfonates / chemistry
  • Nucleic Acid Amplification Techniques*
  • Phenolsulfonphthalein / chemistry
  • Rosaniline Dyes / chemistry

Substances

  • Fluoresceins
  • Fluorescent Dyes
  • Indicators and Reagents
  • Naphthalenesulfonates
  • Rosaniline Dyes
  • malachite green
  • trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate
  • Phenolsulfonphthalein
  • Gentian Violet
  • fluorexon

Supplementary concepts

  • LAMP assay