Ultrafast DNA detection based on turn-back loop primer-accelerated LAMP (TLAMP)

Abstract

Rapid DNA detection is a long-pursuing goal in molecular detection, especially in combating infectious diseases. Loop-mediated isothermal amplification (LAMP) is a robust and prevailing DNA detection method in pathogen detection, which has been drawing broad interest in improving its performance. Herein, we reported a new strategy and developed a new LAMP variant named TLAMP with a superior amplification rate. In this strategy, the turn-back loop primers (TLPs) were devised by ingeniously extending the 5’ end of the original loop primer, which conferred the new role of being the inner primer for TLPs while retaining its original function as the loop primer. In theory, based on the bifunctional TLPs, a total of eight basic dumbbell-like structures and four cyclic amplification pathways were produced to significantly enhance the amplification efficiency of TLAMP. With the enhancing effect of TLPs, TLAMP exhibited a significantly reduced amplification-to-result time compared to the conventional six-primer LAMP (typically 1 h), enabling rapid DNA detection within 20 min. Furthermore, TLAMP proved to be about 10 min faster than the fast LAMP variants reported so far, while still presenting comparable sensitivity and higher repeatability. Finally, TLAMP successfully achieved an ultrafast diagnosis of Monkeypox virus (MPXV), capable of detecting as few as 10 copies (0.67copies/μL) of pseudovirus within 20 min using real-time fluorescence assay or within 30 min using a colorimetric assay, suggesting that the proposed TLAMP offers a sensitive, specific, reliable, and, most importantly, ultrafast DNA detection method when facing the challenges posed by infectious diseases.