A Novel Approach to the Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR Approach
The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcri...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-01-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/22/3/1017 |
| Summary: | The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 10<sup>4</sup> copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization. |
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| ISSN: | 1661-6596 1422-0067 |