DNA快速检测是分子检测领域长期追求的目标,特别是在对抗传染病方面。环介导等温扩增(LAMP)是病原体检测中一种强大且流行的DNA检测方法,这已经引起了人们对提高其性能的广泛兴趣。在这里,我们报道了一种新策略,并开发了一种新的LAMP变异体,命名为TLAMP,具有优异的扩增率.在这个战略中,回转环引物(TLP)是通过巧妙地延伸原始环引物的5'末端设计的,这赋予了作为TLP的内部引物的新角色,同时保留了其作为环引物的原始功能。理论上,基于双功能TLP,共产生了8个基本的哑铃状结构和4个循环扩增通路,显著提高了TLAMP的扩增效率。随着TLP的增强作用,与常规六引物LAMP(通常为1小时)相比,TLAMP显示出显着减少的扩增至结果时间。能够在20分钟内快速检测DNA。此外,TLAMP被证明比迄今为止报道的快速LAMP变体快约10分钟,同时仍具有相当的灵敏度和更高的可重复性。最后,TLAMP成功实现了猴痘病毒(MPXV)的超快诊断,能够使用实时荧光测定法在20分钟内或使用比色测定法在30分钟内检测少至10个拷贝(0.67拷贝/μL)的假病毒,这表明拟议的TLAMP提供了一种敏感的,具体,可靠,and,最重要的是,超快DNA检测方法面临传染病带来的挑战。
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.