Improving Performance of a SARS-CoV-2 RT-LAMP Assay for Use With a Portable Isothermal Fluorimeter: Towards a Point-of-Care Molecular Testing Strategy

Abstract

Frequent and accessible testing is a critical tool to contain the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To develop low-cost rapid tests, many researchers have used reverse transcription loopmediated isothermal amplification (RT-LAMP) with fluorescent readout. Fluorescent LAMP-based assays can be performed using cost-effective, portable, isothermal instruments that are simpler to use and more rugged than polymerase chain reaction (PCR) instruments. However, false-positive results due to nonspecific priming and amplification have been reported for a number of LAMP-based assays. In this report, we implemented a RT-LAMP assay for SARS-CoV-2 on a portable isothermal fluorimeter and a traditional thermocycler; nonspecific amplification was not observed using the thermocycler but did occur frequently with the isothermal fluorimeter. We explored 4 strategies to optimize the SARS-CoV-2 RT-LAMP assay for use with an isothermal fluorimeter and found that overlaying the reaction with mineral oil and including the enzyme Tte UvrD helicase in the reaction eliminated the problem. We anticipate these results and strategies will be relevant for use with a wide range of portable isothermal instruments.

Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing

“…Furthermore, reaction vessels remain closed, and amplicons never need to be manipulated for analysis, reducing the likelihood of environmental crosscontamination [73]. Successful implementation and validation of the RT-LAMP assay on the Axxin T8 has overcome the challenges of adapting assays to point-of-care instruments [36] and shows the potential of the use of this workflow near the point-of-care. FDA Emergency Use Authorization and widespread use of the Lucira COVID-19 All-In-One Test Kit at the time of publication illustrates the potential for similar technologies to be translated for true point-of-care use.…”

“…For RT-LAMP reactions run on the Axxin T8-ISO (T8), assay components described above were doubled to achieve 50 μL reaction volumes with two exceptions [36]: fluorescent dye was used at a 5X concentration instead of 50X [37], and 0.2 μL of Tte UvrD helicase (NEB) was included with each reaction [38,39]. Reactions contained 10 μL of sample unless otherwise noted.…”

Loop-Mediated Isothermal Amplification: From Theory to Practice

“…(1) Mobile thermostats for 0.2-mL tubes with real-time detection of the amplification signal [ 13 , 65 , 120 , 121 ].…”

“…Other enzymes successfully adapted for the LAMP method include the UvrD helicase [ 64 , 65 ], which can reduce the background amplification, as well as uracil-DNA glycosylase [ 66 ], which is used in PCR technology to prevent contamination by amplification products.…”

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.…”