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

Justin C Rolando,Rustem F Ismagilov,Erik Jue,Jacob T Barlow

doi:10.1093/nar/gkaa099

Abstract

Isothermal amplification assays, such as loop-mediated 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.

Highlights

Isothermal methods, such as loop-mediated isothermal amplification (LAMP), are attractive for nucleic acid amplification tests (NAATs) in point-of-care and limited-resource settings [1,2]
We first wished to test whether melting temperature (Tm) could be used to separate specific and non-specific amplification in a LAMP assay run in bulk
We selected a concentration near the limit of detection (LOD) where we might observe both specific and non-specific amplification

Summary

Introduction

Isothermal methods, such as loop-mediated isothermal amplification (LAMP), are attractive for nucleic acid amplification tests (NAATs) in point-of-care and limited-resource settings [1,2]. The ability to optimize LAMP NAATs for a specific target sequence and primer set (specific to a target organism) remains constrained by a limited understanding of how amplification is affected by myriad factors, including polymerase choice, primer design, temperature, time and ion concentrations. Addressing non-specific amplification remains a core problem as it constrains an assay’s limit of detection (LOD). In reactions containing template target molecules, both specific and non-specific amplification reactions may occur. Unlike PCR, LAMP lacks a temperature-gating mechanism, so non-specific reactions consume reagents and compete with specific amplification impacting its kinetics. The presence of non-specific amplicons adversely impacts both the assay’s analytical sensitivity (the fewest template molecules that can be detected) and its analytical specificity (ability to detect the target template in the presence of competing reactions). Classifying reactions as either specific or non-specific amplification would be invaluable both during assay optimization and assay deployment in clinical diagnostics

Methods

Results

Conclusion