Abstract
A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sensitivity and reproducibility to identify the most suitable method(s) for low resource PON applications. A number of methods were found unsuitable as they either involved multiple temperature incubations, were relatively expensive or required relatively large amounts target DNA for amplification. Among the methods examined, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were found to be the most suitable for PON applications as they are both single step methods that provide highly sensitive and reproducible amplifications. The speed of LAMP reactions was greatly enhanced, up to 76%, with the addition of loop primers while the presence of swarm primers and the sequestration of free magnesium ions with nucleotides also enhanced the amplification speed. In contrast, we were unable to enhance RPA’s performance from the original published literature. While both RPA and LAMP have some drawbacks, either isothermal technology can reliably be used for on-site diagnostics with minimal equipment.
Highlights
Disease diagnosis is the critical first step for disease management which is typically performed in modern laboratory facilities by skilled personnel due to the complexity of the techniques involved
Initial selection of isothermal amplification methods with potential PON applications To identify the isothermal technologies that are suitable for PON applications, a large number of isothermal technologies were initially assessed in terms of technical simplicity, cost and speed based on the available literature
strand exchange amplification (SEA) and recombinase polymerase amplification (RPA) use forward and reverse primers similar to those employed in polymerase chain reaction (PCR), to amplify the target of interest (S1 Fig) and utilize a strand displacing DNA polymerase or a combination of recombinase and single stranded DNA binding proteins respectively to enable amplification at a single temperature
Summary
Disease diagnosis is the critical first step for disease management which is typically performed in modern laboratory facilities by skilled personnel due to the complexity of the techniques involved. Testing facilities are often located far away from sampling sites, which results in increased costs incurred by sample transportation as well as delays in disease identification and management. It would be highly beneficial to perform diagnostics at the point-ofneed (PON), such as in a field-based environment [1], as it enables rapid implementation of disease management strategies [2,3,4]. DNA amplification is a powerful platform technology for diagnostics applications due to its speed, sensitivity, specificity, cost-effectiveness and adaptability [5, 6]. The most widely used amplification method is the polymerase chain reaction (PCR) [7] the requirement
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