Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.
Clin Chem
; 67(2): 415-424, 2021 01 30.
Article
in English
| MEDLINE | ID: covidwho-887266
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
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Saliva
/
RNA, Viral
/
Nucleic Acid Amplification Techniques
/
COVID-19 Nucleic Acid Testing
/
SARS-CoV-2
/
COVID-19
Type of study:
Diagnostic study
/
Observational study
/
Prognostic study
Limits:
Humans
Language:
English
Journal:
Clin Chem
Journal subject:
Chemistry, Clinical
Year:
2021
Document Type:
Article
Affiliation country:
Clinchem
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