Helix loop-mediated isothermal amplification of nucleic acids

Abstract

Isothermal nucleic acid amplification has played a key role in the point of care test (POCT). In this study, a helix loop-mediated isothermal amplification (HAMP) method with high specificity, efficiency and rapidity was developed. The MERS-Cov orf1b gene was chosen for the validation and optimization of HAMP. The HAMP analysis was performed at a constant temperature of 61-65 °C and yielded a self-primed spiral structure with no introduction of exogenous gene sequence by two pairs of specially designed primers. The primers for helix loop formation were composed of two complementary primers including the helix forward primer and the helix reverse primer, the 3′ ends of which were complementary to their respective target nucleic acids. HAMP assay can be monitored by fluorescence signals with the addition of Eva Green in the reaction mixture. In addition, an accelerated HAMP was developed after the addition of acceleration probe, which could be finished within 75 min with a sensitivity of 10 copies per reaction. Further, a reverse transcription-HAMP (RT-HAMP) was proven to be feasible for RNA detection by combining the reverse transcriptase with DNA polymerase. Finally, both the HAMP and RT-HAMP assay were visually conducted by using Hydroxynaphthol blue (HNB) as a chromogenic indicator. All in all, it is suggested that the HAMP assay would have great potential in POCT applications.