Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Abstract

Isothermal amplification assays, such as loopmediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

“…However, there is still a challenge to apply it to develop a reliable POC diagnostics for clinical applications due to non-specific signals (e.g., false-positive). 9,10 Recently, RNA-guided CRISPR/Cas nuclease-based nucleic acid detection has shown great promise for the development of next-generation molecular diagnostics technology due to its high sensitivity, specificity and reliability. 11,12 For example, some Cas nucleases (e.g., Cas12a, Cas12b and Cas13a) perform strong collateral cleavage activities in which a crRNA-targetbinding activated Cas can indiscriminately cleave surrounding non-target single-stranded nucleic acids.…”

Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay

“…In recent decades, several isothermal amplification methods, such as recombinase polymerase amplification (RPA) 6 and loop-mediated isothermal amplification (LAMP) 7 , have been developed as attractive alternatives to conventional PCR method because of their simplicity, rapidity and low cost. However, there is still a challenge to apply them to develop accurate and reliable POC testing due to undesired nonspecific amplification signals which may cause false-positive results for SARS-CoV-2 assay 8,9 .…”

Development of a bio-inkjet printed LAMP test kit for detecting human African trypanosomiasis

“…However, some products amplified from a lower concentration of template or no target control (NTC) showed higher temperature peaks (Fig 2B and 2D). These reactions were most probably due to non-specific amplification events involving primer dimers [37]. The Tm values within average ±2 SD were treated as specific amplification.…”