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

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 loop-mediated 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.