Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification. (24th October 2020)

• Record Type: Journal Article
• Authors: Lalli, Matthew A; Langmade, Joshua S; Chen, Xuhua; Fronick, Catrina C; Sawyer, Christopher S; Burcea, Lauren C; Wilkinson, Michael N; Fulton, Robert S; Heinz, Michael; Buchser, William J; Head, Richard D; Mitra, Robi D; Milbrandt, Jeffrey
• Is Part Of: Clinical chemistry. Volume 67:Number 2(2021)
• Page Start: 415
• Page End: 424
• Publication Date: 2020-10-24
• Subjects: SARS-CoV-2 -- COVID-19 -- acute respiratory syndrome -- LAMP -- RT-LAMP -- saliva -- rapid diagnosis -- point-of-care testing -- high-throughput testing -- cost-effective -- preventive medicine; Clinical chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Biochimie -- Périodiques; Diagnostics biologiques -- Périodiques; Biochemistry; Clinical chemistry; Pharmaceutical chemistry; Biochemistry; Laboratory Techniques and Procedures; Klinische chemie; Periodicals; 616.075605
• DOI: 10.1093/clinchem/hvaa267
• Languages: English
• ISSNs: 0009-9147

Abstract

Abstract: Background: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.