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

Clin Chem. 2021 Jan 30;67(2):415-424. doi: 10.1093/clinchem/hvaa267.

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.

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.

MeSH terms

  • COVID-19 / diagnosis*
  • COVID-19 / epidemiology
  • COVID-19 Nucleic Acid Testing / methods*
  • Colorimetry / methods
  • Endopeptidase K / chemistry
  • Humans
  • Limit of Detection
  • Nucleic Acid Amplification Techniques / methods*
  • Pandemics
  • Point-of-Care Testing
  • RNA, Viral / analysis*
  • SARS-CoV-2 / chemistry
  • SARS-CoV-2 / isolation & purification*
  • Saliva / virology*

Substances

  • RNA, Viral
  • Endopeptidase K