Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification

Matthew A. Lalli, Joshua S. Langmade, Xuhua Chen, Catrina C. Fronick, Christopher S. Sawyer, Lauren C. Burcea, Michael N. Wilkinson, Robert S. Fulton, Michael Heinz, William J. Buchser, Richard D. Head, Robi D. Mitra, Jeffrey Milbrandt

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

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.

Original languageEnglish
Pages (from-to)415-424
Number of pages10
JournalClinical chemistry
Volume67
Issue number2
DOIs
StatePublished - Jan 30 2021

Keywords

  • COVID-19
  • LAMP
  • RT-LAMP
  • SARS-CoV-2
  • acute respiratory syndrome
  • cost-effective
  • high-throughput testing
  • point-of-care testing
  • preventive medicine
  • rapid diagnosis
  • saliva

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