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首页> 美国卫生研究院文献>Scientific Reports >Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes
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Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes

机译：使用自猝灭和去猝灭荧光探针实时检测和监测环介导扩增（LAMP）反应

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Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification (iNAAT) technique known for its simplicity, sensitivity and speed. Its low-cost feature has resulted in its wide scale application, especially in low resource settings. The major disadvantage of LAMP is its heavy reliance on indirect detection methods like turbidity and non-specific dyes, which often leads to the detection of false positive results. In the present work, we have developed a direct detection approach, whereby a labelled loop probe quenched in its unbound state, fluoresces only when bound to its target (amplicon). Henceforth, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), it allows for the sequence-specific detection of LAMP amplicons. The FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples. The FLOS-LAMP had a limit of detection of 500 copies of the target with a clinical sensitivity and specificity of 96.8% and 100%, respectively. The high level of specificity is a major advance and solves one of the main shortcomings of the LAMP technology, i.e. false positives. Self-quenching/de-quenching probes were further used with other LAMP primer sets and different fluorophores, thereby demonstrating its versatility and adaptability.
机译：环介导的等温扩增（LAMP）是一种等温核酸扩增（iNAAT）技术，以其简单，灵敏和快速而著称。它的低成本特性使其得以广泛应用，尤其是在资源匮乏的情况下。 LAMP的主​​要缺点是其严重依赖于浊度和非特异性染料等间接检测方法，这常常导致检测出假阳性结果。在目前的工作中，我们已经开发出一种直接检测的方法，在这种方法中，标记的环探针在未结合状态下淬灭，仅在与目标（扩增子）结合时发出荧光。此后称为“自猝灭-LAMP后的环引物荧光”（FLOS-LAMP），它允许对LAMP扩增子进行序列特异性检测。已验证FLOS-LAMP概念可从临床样品中快速检测出人类病原体水痘带状疱疹病毒。 FLOS-LAMP的检测限为500拷贝，临床敏感性和特异性分别为96.8％和100％。高特异性是一大进步，它解决了LAMP技术的主要缺点之一，即假阳性。自淬灭/去淬灭探针还与其他LAMP引物组和不同的荧光团一起使用，从而证明了其多功能性和适应性。

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