Martineau Rhett L, Murray Sarah A, Ci Shufang, Gao Weimin, Chao Shih-Hui, Meldrum Deirdre R
Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University , Tempe, Arizona 85287, United States.
Anal Chem. 2017 Jan 3;89(1):625-632. doi: 10.1021/acs.analchem.6b02578. Epub 2016 Dec 20.
This work describes an enhancement to the loop-mediated isothermal amplification (LAMP) reaction which results in improved performance. Enhancement is achieved by adding a new set of primers to conventional LAMP reactions. These primers are termed "swarm primers" based on their relatively high concentration and their ability to create new amplicons despite the theoretical lack of single-stranded annealing sites. The primers target a region upstream of the FIP/BIP primer recognition sequences on opposite strands, substantially overlapping F1/B1 sites. Thus, despite the addition of a new primer set to an already complex assay, no significant increase in assay complexity is incurred. Swarm priming is presented for three DNA templates: Lambda phage, Synechocystis sp. PCC 6803 rbcL gene, and human HFE. The results of adding swarm primers to conventional LAMP reactions include increased amplification speed, increased indicator contrast, and increased reaction products. For at least one template, minor improvements in assay repeatability are also shown. In addition, swarm priming is shown to be effective at increasing the reaction speed for RNA amplification via RT-LAMP. Collectively, these results suggest that the addition of swarm primers will likely benefit most if not all existing LAMP assays based on state-of-the-art, six-primer reactions.
这项工作描述了对环介导等温扩增（LAMP）反应的一种改进，该改进带来了性能的提升。通过向传统LAMP反应中添加一组新引物来实现这种改进。基于其相对较高的浓度以及尽管理论上缺乏单链退火位点却仍能产生新扩增子的能力，这些引物被称为“群体引物”。这些引物靶向位于相反链上FIP/BIP引物识别序列上游的一个区域，与F1/B1位点大量重叠。因此，尽管在一个已经很复杂的检测中添加了一组新引物，但检测的复杂性并没有显著增加。针对三种DNA模板展示了群体引物引发：λ噬菌体、聚球藻属PCC 6803 rbcL基因和人类HFE。向传统LAMP反应中添加群体引物的结果包括扩增速度提高、指示剂对比度增加以及反应产物增多。对于至少一种模板，还显示出检测重复性有轻微改善。此外，群体引物引发被证明在通过逆转录LAMP（RT-LAMP）提高RNA扩增反应速度方面是有效的。总体而言，这些结果表明，添加群体引物可能会使基于最先进的六引物反应的大多数（如果不是全部）现有LAMP检测受益。
