Kinetics of Isothermal Dumbbell Exponential Amplification: Effects of Mix Composition on LAMP and Its Derivatives.

Loop-mediated isothermal amplification (LAMP) is an exponential amplification method of DNA strands that is more and more used for its high performances. Thanks to its high sensitivity and selectivity, LAMP found numerous applications from the detection of pathogens or viruses through their genome amplification to its incorporation as an amplification strategy in protein or miRNA biomarker quantification. The LAMP method is composed of two stages: the first one consists in the transformation of the DNA strands into dumbbell structures formed of two stems and loops thanks to four primers; then, in the second stage, only two primers are required to amplify the dumbbells exponentially in numerous hairpins of increasing lengths. In this paper, we propose a theoretical framework to analyze the kinetics of the second stage of LAMP, the isothermal dumbbell exponential amplification (IDEA) as function of the physico-chemical parameters of the amplification reaction. Dedicated experiments validate the models. We believe these results may help the optimization of LAMP performances by reducing the number of experiments necessary to find the best parameters.

Development of an Innovative Quantification Assay Based on Aptamer Sandwich and Isothermal Dumbbell Exponential Amplification.

Detecting blood biomarkers such as proteins with high sensitivity and specificity is of the utmost importance for early and reliable disease diagnosis. As molecular probes, aptamers are raising increasing interest for biosensor applications as an alternative to antibodies, which are used in classical enzyme-linked immuno-sorbent assays (ELISA). We have developed a sensitive and antibody-free molecular quantification assay that combines the specificity of aptamers and the sensitivity of the loop-mediated isothermal amplification (LAMP). For the proof-of-concept, we consider two types of biomarkers: (i) a model of oligonucleotide mimicking nucleic acid targets and (ii) the thrombin involved in the complex coagulation cascade as a model protein for which two relevant aptamers form a stable sandwich. The assay protocol is based on a few successive steps, similar to sandwich ELISA. First, aptamer-coated magnetic beads are added to the sample to specifically capture the targets. Then, the sandwich complex is formed by adding the second aptamer. This secondary aptamer is integrated in a larger oligonucleotide dumbbell sequence designed for LAMP detection using only two primers. After a proper rinsing step, the isothermal dumbbell exponential amplification is performed to detect and quantify a low amount of targets (limit of detection ∼ 1 pM for the oligonucleotide and ∼100 pM for thrombin). This study demonstrates that our innovative aptamero-LAMP assay could be relevant for the detection of different types of biomarkers and their quantification at physiological levels. This may also pave the way for antibody-free molecular assays.