Inhibition of Non-specific Amplification in Loop-Mediated Isothermal Amplification via Tetramethylammonium Chloride

Abstract

Loop-mediated isothermal amplification (LAMP) may be used in molecular and point-of-care diagnostics for pathogen detection. The amplification occurs under isothermal conditions using up to six primers. However, non-specific amplification is frequently observed in LAMP. Non-specific amplification has the potential to be triggered by forward and reverse internal primers. And the relatively low reaction temperature (55-65 degrees C) induces the secondary structure via primer-primer interactions. Primer redesign and probe design have been recommended to solve this problem. LAMP primers have strict conditions, such as Tm, GC contents, primer dimer, and distance between primers compared to conventional PCR primers. Probe design requires specialized knowledge to have high specificity for a target. In polymerase chain reaction (PCR), some chemicals or proteins are used for improving specificity and efficiency. Therefore, we hypothesized that additives can suppress the non-specific amplification. In this study, tetramethylammonium chloride (TMAC), formamide, dimethyl sulfoxide, Tween 20, and bovine serum albumin have been used as LAMP additives. In our study, TMAC was presented as a promising additive for suppressing non-specific amplification in LAMP.