One advantage of iNAAT’s over PCR is that they amplify their targets at a constant temperature, thereby obviating the time consuming thermocycling steps required for any two- or three-step PCR. Examples of such technologies include: strand-displacement amplification (SDA) 4, helicase-dependent amplification (HDA) 5, recombinase polymerase amplification (RPA) 6 and loop-mediated amplification (LAMP) 7. Self-quenching/de-quenching probes were further used with other LAMP primer sets and different fluorophores, thereby demonstrating its versatility and adaptability.Ĭhallenges in exploiting polymerase chain reaction (PCR) for point-of-care (POC) diagnostics have resulted in the development of nucleic acid amplification tests based on isothermal principles that are collectively referred to as iNAAT’s 1, 2, 3. The high level of specificity is a major advance and solves one of the main shortcomings of the LAMP technology, i.e. 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 FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples. Henceforth, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), it allows for the sequence-specific detection of LAMP amplicons.
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). 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. Its low-cost feature has resulted in its wide scale application, especially in low resource settings. Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification (iNAAT) technique known for its simplicity, sensitivity and speed.
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