Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA.
Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA.
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DOI：
10.1039/c7an01897e
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发表时间：
2018-04-16
期刊：
影响因子：
--
通讯作者：
Wang E
中科院分区：
文献类型：
--
作者：
Meagher RJ ;Priye A ;Light YK ;Huang C ;Wang E
环介导的等温扩增（LAMP）与逆转录（RT）相结合，由于在护理点或低资源环境中使用的几个理想特性，已成为一种流行的病毒RNA检测技术。LAMP中大量的引物（每个靶标6个）导致引物二聚体相互作用的可能性增加，特别是内部引物由于其长度（通常为40-45个碱基）而易于形成稳定的发夹结构。虽然引物二聚体和发夹结构是已知的核酸扩增技术中要避免的特征，但文献中关于这些结构对LAMP或RT-LAMP检测的影响的定量信息很少。在这项研究中，我们研究了引物二聚体和发夹对先前发表的登革热病毒和黄热病病毒引物集的影响。我们证明，当使用插层染料实时监测和使用QUASR技术监测荧光终点时，对引物进行微小的改变以消除可扩增的引物二聚体和发夹，可以提高分析的性能。我们还讨论了这些微小变化对可扩增二级结构整体稳定性的热力学影响，并提出了一个单一的热力学参数，该参数可以与LAMP引物相关的非特异性扩增概率相关。基于热力学预测的RT-LAMP启动位点的碰撞可以显著减少非特异性背景扩增。
Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40–45 bases). Although primer dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter that can be correlated to the probability of non-specific amplification associated with LAMP primers. Bumping the priming sites for RT-LAMP based on thermodynamic predictions can dramatically reduce non-specific background amplification.
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