Abstract：
Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5\' end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.
摘要：
基于荧光染料的环介导等温扩增(LAMP)是一种灵敏的核酸检测方法,但仅限于单重检测，可能产生非特异性信号。在这项研究中,我们提出了一种基于双功能探针的实时LAMP扩增方法，用于单路或多路检测。双功能探针是通过修饰荧光团的5'末端和LAMP引物之一上的内部猝灭剂而衍生的；因此，它可以同时参与LAMP过程和信号放大。在将双功能探针掺入双链DNA扩增子期间,荧光强度经历累积指数增加。基于双功能探针的LAMP方法简化且具有成本效益，因为引物设计和实验操作与普通LAMP完全一致。与其他基于当前探针的方法不同，这种方法不需要额外的酶，序列，或特殊的探头结构。此外，它比其他几种基于探针的LAMP方法快10分钟。基于双功能探针的LAMP方法允许在一锅反应中同时检测目标副溶血性弧菌DNA和内部扩增对照，展示了其多路检测的潜力。
