Classification of Multiple DNA Dyes Based on Inhibition Effects on Real-Time Loop-Mediated Isothermal Amplification (LAMP): Prospect for Point of Care Setting.

Than Linh Quyen,Mogens Madsen,Dang Duong Bang,Anders Wolff,Tien Anh Ngo

doi:10.3389/fmicb.2019.02234

Abstract

LAMP has received great interest and is widely utilized in life sciences for nucleic acid analysis. To monitor a real-time LAMP assay, a fluorescence DNA dye is an indispensable component and therefore the selection of a suitable dye for real-time LAMP is a need. To aid this selection, we investigated the inhibition effects of twenty-three DNA dyes on real-time LAMP. Threshold time (Tt) values of each real-time LAMP were determined and used as an indicator of the inhibition effect. Based on the inhibition effects, the dyes were classified into four groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect. The signal to noise ratio (SNR) and the limit of detection (LOD) of the dyes in groups 1, 2, and 3 were further investigated, and possible inhibition mechanisms of the DNA dyes on the real-time LAMP are suggested and discussed. Furthermore, a comparison of SYTO 9 in different LAMP reactions and different systems is presented. Of the 23 dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow were the best dyes with no inhibitory effect, low LOD and high SNR in the real-time LAMP reactions. The present classification of the dyes will simplify the selection of fluorescence dye for real-time LAMP assays in point of care setting.

Highlights

Over the last 18 years, LAMP has been used widely in the laboratory and in point of care (POC) settings to analyze nucleic acid as well as to detect pathogens (Notomi et al, 2000; Njiru, 2012; Seki et al, 2018)
It has been shown that DNA dyes such as SYTO 82 could be used for real-time LAMP using a lowcost charge-coupled device (CCD) based fluorescence imaging system that could be comparable to a commercial real-time PCR instrument (Ahmad et al, 2011)
We thoroughly investigated the inhibitory effects and classified 23 DNA dyes that span a wide range of four groups of dyes with different optical properties in a real-time LAMP assay for detection of Salmonella Enteritidis

Summary

Introduction

Over the last 18 years, LAMP has been used widely in the laboratory and in point of care (POC) settings to analyze nucleic acid as well as to detect pathogens (Notomi et al, 2000; Njiru, 2012; Seki et al, 2018). Real-Time LAMP Using DNA Dyes considered an attractive method for POC systems (de Paz et al, 2014; Sun et al, 2015). The use of DNA dyes is considered an excellent alternative detection method in real-time LAMP since it is simple and it can enhance the sensitivity of the assay as compared to the turbidity measurement (Cao et al, 2015). Sun et al (2015) reported the use of SYTO 62 for real-time LAMP in an integrated LOC system. Those reports showed the potential of using DNA dyes in POC devices

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Results

Conclusion