Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
Sci Rep. 2017 Jan 6;7:40125. doi: 10.1038/srep40125.
This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25-45 bp, beacon concentration of 0.6-1 pmol/μL, and reaction temperature of 60-65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.
本研究建立了一种恒温荧光定量检测方法，将环介导等温扩增（LAMP）与分子信标相结合。LAMP 的优点是方便、高效，因为它不需要热循环仪，并且结果可以通过肉眼轻松可视化。然而，目前 LAMP 技术的一个主要缺点是使用间接评估方法（例如电泳、SYBR Green I 染料、沉淀、羟基萘酚蓝染料、浊度法、钙黄绿素/Mn 染料和复合探针法），这些方法无法区分所需产物和非特异性扩增产物，从而导致假阳性。分子信标可避免此问题，因为只有当与靶 DNA 结合时，分子信标才会产生荧光信号，因此可作为扩增产物的直接指示物。我们的分析确定了分子信标作为 LAMP 评估工具的最佳条件：信标长度为 25-45bp，信标浓度为 0.6-1pmol/μL，反应温度为 60-65°C。总之，我们验证了一种新型的分子信标环介导等温扩增方法（MB-LAMP），实现了 LAMP 产物的直接检测。
