RT-Lamp Assay Combining Multi-Fluorescent Probes for SARS-CoV-2 RNA Detection and Variant Differentiation

Simple and accurate testing tools for SARS-CoV-2 viral RNA detection are essential for the prevention of the spread of the virus and timely governmental actions. While the reverse transcription quantitative polymerase chain reaction (RT-qPCR) that requires a sophisticated thermocycler is the gold standard for viral RNA detection, isothermal amplification-based detection might be ideal for screening COVID-19. Herein, we present a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of ORF1ab and N gene fragments of SARS-CoV-2. Positive results were obtained using two primer sets for SARS-CoV-2 with a limit of detection of 20 and 2 copies/μL for ORF1ab and N gene fragments, respectively. Additionally, the RT-LAMP based assay can be used to detect single-site differences in target genes using two one-step displacement (OSD) probes targeting wild-type and mutant S genes. The fluorescence intensity of the OSD probe is only related to the template sequence, rather than the concentration of the templates. Our method has high sensitivity for quantification and high specificity for mutation differentiation. The proposed method does not require sophisticated thermal cycler instrumentation and can be used in clinical settings in resource-limited regions.