4.7 Article

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

期刊

出版社

MDPI
DOI: 10.3390/ijms22031017

关键词

SARS-CoV-2; accurate and rapid identification; RT-LAMP-BART; digital PCR system; digital RT-LAMP approach

资金

  1. National Natural Science Foundation of China [61971123, 61571114]

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The study developed an improved and sensitive approach to SARS-CoV-2 detection using BART and RT-LAMP protocols, which was also compatible with a digital LAMP system. By improving RNA availability in samples and utilizing a simulated digital RT-LAMP approach, real-time detection with a lower limit of detection was achieved, along with enhancements in the overall dynamic range of the assay system.
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(4) 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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