Jiang Yu Sherry, Bhadra Sanchita, Li Bingling, Wu Yuefeng Rose, Milligan John N, Ellington Andrew D
†Center for Systems and Synthetic Biology, ‡Department of Chemistry, §Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, United States.
Anal Chem. 2015 Mar 17;87(6):3314-20. doi: 10.1021/ac504387c. Epub 2015 Mar 2.
Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.
DNA的环介导等温扩增（LAMP）是一种强大的等温核酸扩增方法，能在一小时内从少于100份模板DNA中产生超过10⁹份拷贝。遗憾的是，尽管扩增反应极为强大，但对LAMP产物进行实时且特异性检测在分析上仍具有挑战性。为了提高LAMP检测的特异性，并使读数更简单可靠，我们用一种称为一步链置换（OSD）的由链置换介导的链交换反应取代了通常用于实时荧光监测的嵌入染料。由于链置换介导的链交换具有固有的序列特异性，OSD报告分子能够成功区分副产物与对应于生物医学相关的结核分枝杆菌RNA聚合酶（rpoB）和黑色素瘤相关生物标志物BRAF的模板产生的真正扩增子。OSD能够在合成痰液等复杂混合物中对rpoB进行是/否检测，并且在野生型基因含量多出20倍的情况下，对突变型BRAF等位基因（V600E）的是/否检测中也显示出单核苷酸特异性。在多重LAMP反应中对不同基因进行实时检测也被证明是可行的。开发用于等温扩增反应的简单、易于设计的模块化TaqMan探针类似物通常应能提高这些反应的适用性，并最终可能有助于即时检测的发展。
