Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Abstract

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loopmediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Allele-Specific Recombinase Polymerase Amplification to Detect Sickle Cell Disease in Low-Resource Settings

“…Finally, the diluted lysate from a normal volunteer was tested in 25 μL reactions on a miniature, low-cost, open-source fluorimeter designed to be used near the point of care with any conventional heat block Figure E shows clear amplification in the β A allele reaction only, correctly genotyping this sample as AA.…”

A sensitive and simple RT-LAMP assay for sarbecovirus screening in bats

“…The fluorescence measurement can be achieved using a simple portable fluorometer, which can cost as low as approximately USD 60, or a commercial one with a cost of approximately USD 1,500 ( 25 ). Moreover, in order to perform real-time result reading and quantitative analysis of the viral titers of the samples by comparing time to positivity, the fluorescence can be measured in real time by placing the fluorometer atop the RT-LAMP assay heat block by enclosing in a compact 3D printed housing ( 26 ). Alternatively, there is also a newly developed molecular detection system, eGGi, specifically designed for on-site testing based on the isothermal amplification method ( 27 ).…”

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection

“…Therefore, the facility for the probe-based LAMP methods is more likely to be more costly than the traditional LAMP methods. It is worth noting that some portable fluorescence analyzers and isothermal fluorimeters with minimal laboratory resources are currently available or under development (Supplementary Table S1) [70][71][72][73][74][75]. Furthermore, end-point fluorescence detection requires much less instrumentation than real-time monitoring.…”

“…Second, the results of the probe-based LAMP methods can be real-time monitored using a real-time (thermal or isothermal) fluorimeter or be observed at the end-point using a regular fluorimeter [70,71,73]. A small, portable, cheap, and battery-powered isothermal fluorimeter should be developed.…”