Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification
© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissionsoup.com..
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.
| Errataetall: | |
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| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:67 |
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| Enthalten in: |
Clinical chemistry - 67(2021), 2 vom: 30. Jan., Seite 415-424 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Lalli, Matthew A [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 02.03.2021 Date Revised 30.03.2024 published: Print UpdateOf: medRxiv. 2020 May 11;:. - PMID 32511508 Citation Status MEDLINE |
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| doi: |
10.1093/clinchem/hvaa267 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 500 | |a Date Revised 30.03.2024 | ||
| 500 | |a published: Print | ||
| 500 | |a UpdateOf: medRxiv. 2020 May 11;:. - PMID 32511508 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissionsoup.com. | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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% | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
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| 700 | 1 | |a Chen, Xuhua |e verfasserin |4 aut | |
| 700 | 1 | |a Fronick, Catrina C |e verfasserin |4 aut | |
| 700 | 1 | |a Sawyer, Christopher S |e verfasserin |4 aut | |
| 700 | 1 | |a Burcea, Lauren C |e verfasserin |4 aut | |
| 700 | 1 | |a Wilkinson, Michael N |e verfasserin |4 aut | |
| 700 | 1 | |a Fulton, Robert S |e verfasserin |4 aut | |
| 700 | 1 | |a Heinz, Michael |e verfasserin |4 aut | |
| 700 | 1 | |a Buchser, William J |e verfasserin |4 aut | |
| 700 | 1 | |a Head, Richard D |e verfasserin |4 aut | |
| 700 | 1 | |a Mitra, Robi D |e verfasserin |4 aut | |
| 700 | 1 | |a Milbrandt, Jeffrey |e verfasserin |4 aut | |
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