A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3–4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

中文翻译：

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使用新型带万能报道基因的介体置换探针，对循环介导的等温扩增进行简化的实时多重检测

基于DNA扩增过程中荧光强度的变化，多种用于环介导的等温扩增（LAMP）的实时检测技术可同时检测多个靶标。但是，这些技术取决于包含靶标特异性序列的荧光探针。这使对不同目标的适应变得复杂，导致耗时的测定优化。在这里，我们介绍了第一个用于多路复用LAMP的通用实时检测技术。新颖的方法允许简单的测定设计，并且易于针对各种靶标实施。这项创新功能包括一个介体位移探针和一个通用报告器。在靶DNA的扩增过程中，将介体从介体置换探针置换。然后，它与报道分子杂交，产生荧光信号。n = 36），改善了信噪比的荧光比（MD：5.9±0.4，MB：2.7±0.4；n = 15），并显示出同样好或更好的分析性能参数。在不同的多重测定一个万向介体-报告基因组的可用性已成功证实了HIV-1的二重RT-LAMP和HTLV-1和的二重LAMP杜克雷嗜血杆菌和梅毒螺旋体，靶浓度和时间之间的两个示出良好的相关性积极。由于其简单的实现方式，建议将通用介体-报告器集的使用扩展到检测其他各种诊断面板。