Real-Time, Digital LAMP with Commercial Microfluidic Chips Reveals the Interplay of Efficiency, Speed, and Background Amplification as a Function of Reaction Temperature and Time

Abstract

Real-time, isothermal, digital nucleic acid amplification is emerging as an attractive approach for a multitude of applications including diagnostics, mechanistic studies, and assay optimization. Unfortunately, there is no commercially available and affordable real-time, digital instrument validated for isothermal amplification; thus, most researchers have not been able to apply digital, real-time approaches to isothermal amplification. Here, we generate an approach to real-time digital loop-mediated isothermal amplification (LAMP) using commercially available microfluidic chips and reagents and open-source components. We demonstrate this approach by testing variables that influence LAMP reaction speed and the probability of detection. By analyzing the interplay of amplification efficiency, background, and speed of amplification, this real-time digital method enabled us to test enzymatic performance over a range of temperatures, generating high-precision kinetic and end-point measurements. We were able to identify the unique optimal temperature for two polymerase enzymes while accounting for amplification efficiency, nonspecific background, and time to threshold. We validated this digital LAMP assay and pipeline by performing a phenotypic antibiotic susceptibility test on 17 archived clinical urine samples from patients diagnosed with urinary tract infections. We provide all the necessary workflows to perform digital LAMP using standard laboratory equipment and commercially available materials. This real-time digital approach will be useful to others in the future to understand the fundamentals of isothermal chemistries, including which components determine amplification fate, reaction speed, and enzymatic performance. Researchers can also adapt this pipeline, which uses only standard equipment and commercial components, to quickly study and optimize assays using precise, real-time digital quantification, accelerating development of critically needed diagnostics.

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

“…We found that TTP can be heterogeneous while T m is constant (28.6 ± 8.9 min with 87.5 ± 0.2 • C), indicating that the same product may initiate at different times ( Figure 5F). This is consistent with our knowledge of the stochastic initiation of LAMP (23,(38)(39). Further, we observed some variability in the maximum rate despite similar T m (23.7 ± 6.8 RFU/30 s, with 87.5 ± 0.2 • C T m ), which indicates the same product may amplify at different velocities ( Figure 5G).…”

“….75 • C ( Figure 6E) and we observed better separation of specific and non-specific amplification than with Bst 2.0 ( Figure 6F and Supplementary Figure S8B). Both enzymes had highly variable TTP, which we have observed previously (23) and attribute to stochastic initiation of LAMP. Bst 2.0 had both earlier specific amplification and later non-specific amplification than Bst 3.0.…”

“…In these experiments, we chose to use a microscope, instead of the custom real-time amplification instrument we used previously (23,24), because the microscope has superior optical properties (greater pixels per partition and lower exposure time requirements) to access higher temporal resolution and enhanced kinetic measurements.…”

“…Digital single-molecule methods separating individual amplification events into discrete compartments, eliminating interference among individual amplification events (21,22). Furthermore, digital experiments consist of thousands of reactions that run in parallel and thus provide valuable statistical information (21)(22)(23). We used real-time imaging to monitor the kinetics of 20,000 dLAMP reactions per experiment and observe ∼1.2 × 10 6 reactions in total.…”

Microfluidic Rapid and Autonomous Analytical Device (microRAAD) to Detect HIV from Whole Blood Samples

“…One such isothermal amplification method, loop-mediated isothermal amplification (LAMP), provides specific and efficient amplification of target nucleic acids by targeting 8 unique sequences (Nagamine et al, 2002). Operable at a single temperature (most efficiently between 65 and 72 °C) (NEB, n.d.; Rolando et al, 2019), LAMP robustly amplifies even in the presence of complex sample matrices, further reducing sample processing and instrumentation requirements (Clayton et al, 2019;Mori and Notomi, 2009;Phillips et al, 2018). To expedite sample preparation steps, such as reverse transcription (RT) of HIV RNA targets, several groups have demonstrated that RT can be performed using the same assay conditions as LAMP (Curtis et al, 2012;Damhorst et al, 2015;Odari et al, 2015;Rudolph et al, 2015).…”