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.
Highlights
Amongst the isothermal nucleic acid amplification (iNAAT)’s, the Loop-mediated isothermal amplification (LAMP) technology per se, has found wide scale application in both laboratory and POC diagnostics[11]
LAMP is a DNA amplification technique that is attractive for molecular diagnostic assays, especially for POC use[11]
Like other isothermal amplification systems, LAMP is plagued by false positive signals being detected in negative control reactions[29], which has hampered its use in clinical diagnostics
Summary
Amongst the iNAAT’s, the LAMP technology per se, has found wide scale application in both laboratory and POC diagnostics[11]. Fluorophores labelled nucleic acids, that hybridize, in a sequence dependent manner, to a transiently generated single-stranded DNA structure, have proven to be an ideal solution to any non-specific, TM dye-based detection system Examples of such include the, hydrolysis-based TaqMan probes developed for qPCR18 and molecular beacons[19] among host of others. We demonstrate the use of self-quenching fluorogenic probes as an alternative approach to detect and monitor LAMP reactions in real-time These probes, originally developed as part of luminescence upon extension technology[26,27], were tested for their suitability to detect LAMP reaction products in real-time. This new real-time LAMP technology, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), was optimized to detect a clinically important human pathogen, Varicella-zoster virus (VZV)
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