Quantitative comparison of isothermal DNA amplification methods
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In the field of isothermal DNA amplification, four of the widest used methods are recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), isothermal and chimeric primer initiated amplification of nucleic acids (ICAN) and loop-mediated isothermal amplification (LAMP). The areas of comparison between the four methods include robustness, limit of detection, specificity, ease of primer design, and reaction time. LAMP, developed in 2000 by Tsugunori Notomi et. al, has many characteristics that render it extremely attractive for therapeutic applications, clinical diagnosis, and easy DNA amplification. The presence of target DNA can be easily detected by a visual turbidity test, which can be a crucial advantage in a clinical diagnosis setting. Various primer sets, designed with the Primer Explore program (version 4), that target known genes of malaria and tuberculosis are tested in this project. Preliminary and quantitative real time experiments with primer sets for Microbacterium tuberculosis RPOB, Plasmodium falciparum CytB, and falciparum, ovale, malariae, and vivax 18S, using the standard LAMP protocol have yielded positive results. Similar analyses and experiments on the other three isothermal methods will reveal pros and cons of each technique, assisting others in making an educated decision on which method best suits their needs.