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.
Highlights
I n this paper, we describe a methodology to use commercially available chips, reagents, and microscopes to perform real-time digital loopmediated isothermal amplification (LAMP)
Despite the value that realtime digital LAMP (dLAMP) can bring to diagnostics, this method is difficult to implement, especially for those without a background in microelectromechanical systems or microfluidics, because there is no commercial system for real-time, digital isothermal amplification
The chips consist of an array of 20000 uniform partitions (Figure 1), each 60 μm in diameter and an estimated 0.75 nL internal volume, which is similar to the volumes typically used in dLAMP.[16,18,20−23,25,26,28] These chips are loaded by pipetting the sample mixture into the plastic “blade” provided with the chips and dragging the blade at a 70−80° angle to the chip to load the sample mixture by capillarity
Summary
I n this paper, we describe a methodology to use commercially available chips, reagents, and microscopes to perform real-time digital LAMP. LAMP and other isothermal technologies are fast and sensitive, but when performed in a bulk format in microliter volumes (e.g., in a tube), they provide only semiquantitative (log-scale) resolution or presence/absence measurements.[7−15] As a result, when optimizing an assay, it is difficult to quantify how small changes in assay conditions (e.g., in primers, reagents, or temperature) impact the reaction’s speed and analytical sensitivity To reliably understand these effects with high precision would require hundreds of bulk experiments per condition.[16] For the field to be able to take full advantage of the capabilities of LAMP, researchers need to be able to optimize reaction conditions by understanding and testing the variables that may influence reaction speed and probability of detection. Those who would most benefit from optimized digital isothermal reactions (e.g., those working on POC diagnostics) cannot efficiently improve them
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