Ultimate Single-Copy DNA Detection Using Real-Time Electrochemical LAMP

Abstract

Herein, we report the first successful detection of single DNA copies using real-time electrochemical loop-mediated isothermal amplification (LAMP). Toward this end, real-time electrochemical LAMP amplifications of bacteriophage M13mp18 DNA and of a genomic DNA sequence from the pathogen Flavobacterium columnare were systematically run each with four different redox reporters (i.e., three intercalating reporters with different ds-DNA binding strengths and one nonintercalating probe able to sense the pyrophosphate ion concentration produced during LAMP). In order to provide a reliable, rapid assessment of the LAMP performances, the experiments were carried out using a prototype high-throughput house-built automatized electrochemical read-out device, optimized for parallel monitoring of up to 48 samples. The electrochemical results were then compared to those achieved with conventional real-time fluorescence LAMP and PCR methods (using commercially available fluorescent dyes and standard benchtop equipment)...