Gold nanoparticle-mediated nucleic acid isothermal amplification with enhanced specificity.

Loop-mediated isothermal amplification is a promising method in the area of nucleic acid detection. However, it suffers from a high rate of false-positive amplifications that largely restrict its application. In this study, we observed gold nanoparticles (AuNP) absorbing single-stranded DNA primers and interacting with Bst DNA polymerase via electrostatic adsorption. As a result of these interactions, the presence of the gold nanoparticles exerted a hot-start effect on the loop-mediated isothermal amplification system. Based on these results, we developed a novel AuNP-mediated nucleic acid isothermal amplification assay. This assay displays significantly enhanced specificity-the proportion of false positive decreased from 76% to 0% and from 100% to 0% for the detection of rotavirus and the β-actin gene, respectively, with the hot-start temperature of 48 °C. Moreover, our AuNP-based assay maintained good sensitivity and a satisfactory detection limit (1 × 103copies/μL) compared with the conventional assay. This approach has the potential to solve the nonspecificity problem of loop-mediated isothermal amplification, thereby promoting its real-world application, particularly, in clinical settings.