Abstract
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. 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 in one pot. Using two primer sets and two molecular beacon (MB) probes respectively labelled with different fluorophore, positive results were obtained with a limit of detection of 20 and 2 copies/μL for ORF1ab and N gene fragments, respectively. Moreover, the RT-LAMP based assay was applied to detect single-site differences in S genes using two one-step displacement (OSD) probes targeting wild-type and mutant (P681R mutation was chosen as model) genes. Through that, the wild type strain and P681R mutant variant were well distinguished from each other, and a preliminary observation was also made on other mutations at this site such as P681H. The proposed method has high sensitivity for quantification and high specificity for mutation differentiation. In addition, it does not require accurate sophisticated thermal cycler instrumentation and can be used in clinical settings in resource-limited regions.
Keywords: Fluorescent probes; Reverse transcription loop-mediated isothermal amplification (RT-LAMP); SARS-CoV-2 RNA detection; Variant differentiation.
【저자키워드】 Reverse transcription loop-mediated isothermal amplification (RT-LAMP), Fluorescent probes, SARS-CoV-2 RNA detection, Variant differentiation., 【초록키워드】 SARS-CoV-2, Mutation, Transcription, variant, virus, Spread, RT-LAMP, sensitivity, specificity, Loop-mediated isothermal amplification, limit of detection, reverse transcription, isothermal amplification, SARS-CoV-2 RNA, mutant, molecular, N gene, P681H, P681R, quantification, wild type, S gene, Fluorescent probes, SARS-CoV-2 RNA detection, SARS-CoV-2 viral RNA, ORF1ab, type strain, observation, wild type strain, positive result, clinical settings, wild-type, clinical setting, ORF1, viral RNA detection, simple, probe, Genes, regions, detect, primer set, addition, applied, can be used, 【제목키워드】 variant, probe,