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 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 <i>Tte</i> 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.