Isothermal amplification using sequence-specific fluorescence detection of SARS coronavirus 2 and variants in nasal swabs

• Published in: BioTechniques Vol. 72; no. 6; pp. 263 - 272
• Main Authors: Jones, Les, Naikare, Hemant K, Mosley, Yung-Yi C, Tripp, Ralph A
• Format: Journal Article
• Language: English
• Published: England Future Science Ltd 01.06.2022; Taylor & Francis Group
• Subjects: diagnostics; fluorescence; FQ-LAMP; POC; RT-LAMP; SARS coronavirus 2; variant; FQ-LAMP; RT-LAMP; diagnostics; POC; fluorescence; variant; SARS coronavirus 2
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/btn-2022-0037

Coronavirus disease 2019 is a public health challenge requiring rapid testing for the detection of infections and transmission. Nucleic acid amplification tests targeting SARS coronavirus 2 (CoV2) are used to detect CoV2 in clinical samples. Real-time reverse transcription quantitative PCR is the standard nucleic acid amplification test for CoV2, although reverse transcription loop-mediated isothermal amplification is used in diagnostics. The authors demonstrate a sequence-specific reverse transcription loop-mediated isothermal amplification-based nucleic acid amplification assay that is finished within 30 min using minimally processed clinical nasal swab samples and describe a fluorescence-quenched reverse transcription loop-mediated isothermal amplification assay using labeled primers and a quencher oligonucleotide. This assay can achieve rapid (30 min) and sensitive (1000 plaque-forming units/ml) fluorescence detection of CoV2 (WA1/2020), B.1.1.7 (Alpha) and variants of concern Delta (B.1.617.2) and Omicron (B.1.1.529) in nasal samples. The authors describe a sequence-specific nucleic acid amplification assay (fluorescence-quenched reverse transcription loop-mediated isothermal amplification) based on a modified reverse transcription loop-mediated isothermal amplification assay that utilizes a fluorescence-labeled reporter primer and a short complementary oligonucleotide quencher to detect SARS coronavirus 2 in minimally processed clinical nasal swab samples. The fluorescence-quenched reverse transcription loop-mediated isothermal amplification assay is completed in 30 min without purifying RNA and achieves reproducible, sensitive and specific (1000 plaque-forming units/ml) detection of SARS coronavirus 2 WA1/2020 and three SARS coronavirus 2 variant viruses while not signaling on three closely related human coronaviruses or two other heterologous human respiratory viruses.