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

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). The best electrochemical performances were obtained with the two strongest intercalating redox reporters, affording analytical sensitivity and detection limits comparable to real-time fluorescence LAMP. These electrochemical reporters enabled us to assemble highly accurate, log linear calibration curves in only 30 min, over a six-decade dynamic concentration range with detection limits as low as two DNA copies of genomic target in 50 mu L. These are to our knowledge the best results so far reported for electrochemical-based real-time LAMP, rivaling those achieved with fluorescence-based real-time LAMPs. The major advantages of our electrochemical LAMP approach over the fluorescence-based LAMP method include miniaturization, portability, robustness, low cost, and the ability to work with nontransparent reaction mixtures.