Diverse methods of reducing and confirming false-positive results of loop-mediated isothermal amplification assays: A review

Abstract

Loop-mediated isothermal amplification (LAMP), a rapid and sensitive isothermal nucleic acid amplification method, is a promising alternative to other molecular amplification techniques due to its superior specificity and sensitivity. However, due to primer dimerization, LAMP results in nonspecific and nontemplate amplification. And during the amplification confirmation process, there is carry-over contamination. These factors can result in false-positive results that overestimate the amount of DNA, preventing accurate detection. This review outlined several techniques for reducing false-positive LAMP results before amplification and confirming false-positive results after amplification. Before the amplification step, DNA polymerase activity can be decreased with organic additives such as dimethyl sulfoxide, betaine, and pullulan to prevent nonspecific amplification. The enzyme uracil-DNA-glycosylase (UDG) can eliminate false-positive results caused by carry-over contamination, and the hot-start effect with gold nanoparticles can reduce nonspecific amplification. When confirming false-positive results using clustered regularly interspaced short palindromic repeats, guide RNA accurately detects LAMP amplification, allowing differentiation from nonspecific amplification. By confirming amplification, the colorimetric change in the deoxyribozyme (DNAzyme) formed by the reaction of the G-quadruplex sequence of the LAMP amplicon and hemin can distinguish false-positive results. Lateral flow immunoassay can distinguish false-positive results by accurately recognizing hybridized probes to LAMP amplicons.