Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes

Abstract

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.

Figure 1

Fluorescence signal from VZV62 dT-FAM probes. (A) Signal profile obtained after converting the Outer (VZV62F3), Inner (VZV62FIP) and Loop (VZV62LPB) primers into dT-FAM fluorescent probes. Input template concentration pVZV-ORF62 plasmid: 1 × 10⁶ copies/µL. (B) Variation of fluorescence signal intensity due to varying levels of the Base Mix ranging from 0.5X, 1.0X, 1.5X, 2.0X and 2.5X. Input template concentration pVZV-ORF62 plasmid: 1 × 10⁶ copies/µL. Water was used as a negative control. Each Time[30 sec interval] unit (X-axis) represents 30 seconds on the ESEQuant instrument.

Figure 2

Optimization of signal intensity using the VZV62LPB-FAM probe. (A) Effect of increasing levels of F3/B3 primer pair from 0.5 µM (Mix 5), 1.0 µM (Mix 6) and 2.0 µM (Mix 7_OPT). (B) Variation in signal intensity with increasing levels of the VZV62LPB-FAM probe from 0.1 µM, 0.2 µM and 0.3 µM. Input template concentration of pVZV-ORF62 plasmid: 1 × 10⁶ copies/µL. Each Time[30 sec interval] unit (X-axis) represents 30 seconds on the ESEQuant instrument.

Figure 3

Detection of VZV from DFA positive clinical samples using VZV62 FLOS-LAMP assay. VZV DFA-positive clinical samples detected by the VZV62 FLOS-LAMP assay when (A) clinical samples were added directly into the reaction mixture and (B) after rapid DNA extraction using InstaGene™ matrix. Each Time[30 sec interval] unit (X-axis) represents 30 seconds on the ESEQuant instrument.

Figure 4

Schematic depiction of the binding of VZV62LPB-FAM probe to its target. The FAM fluorophore attached to the VZV62LPB-FAM probe is self-quenched in unbound state. Post binding to the dumbbell shaped DNA target, the FAM fluorophore is de-quenched resulting in fluorescence development. For simplicity, only one dumbbell which would bind to the LPB loop primer (VZV62LPB) is shown in this depiction.