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

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.

Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Accurate Confirmatory Diagnosis of HTLV-1/2 Infection

“…LAMP has the advantage of being carried out in a conventional thermocycler, or even in a water bath or thermal block. In addition, LAMP results can also be revealed by SYBR Green I by pre-loading the dye in the inner side of microtube caps before the reaction, since it inhibits DNA amplification by Bst DNA polymerases [7], or using DNA dyes such as SYTO9, SYTO13 and SYTO16 directly in the reaction mixture [71]. This abrogates the need for opening tubes after the reaction, thus minimizing the chance of amplicon contamination.…”

Simple and Visible Detection of Novel Astroviruses Causing Fatal Gout in Goslings Using One-Step Reverse Transcription Polymerase Spiral Reaction Method

“…This means the reaction must be uncovered in order to add the dye, increasing the risk of aerosol cross-contamination. SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow—different from SYBR Green I, —were found to be the best dyes with no inhibitory effect in the real-time LAMP reactions ( 22 ). Whether these dyes in high concentration could be used for visible observation by the naked eye under natural light needs further evaluation.…”

Point-of-care diagnostics for infectious diseases: From methods to devices

“…To circumvent the complex heating and cooling control in conventional PCR devices and increase the detection speed, a series of isothermal nucleic acid amplification methods have been developed for POC diagnostic applications, such as rolling circle amplification (RCA) [459] , [460] , [461] , LAMP [354] , [462] , [463] , [464] , RPA [465] , [466] , strand displacement amplification (SDA) [467] , [468] , helicase dependent amplification (HDA) [469] , NASBA [355] , multiple cross displacement amplification [470] , [471] , [472] and so on. These isothermal nucleic acid amplification methods were conducted at a fixed temperature without complex thermal cycling equipment, making them more applicable for POC diagnostics [294] , [442] , [473] , [474] .…”