Abstract
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 104 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.
Highlights
COVID-19 is a pandemic disease caused by SARS-CoV-2(severe acute respiratory syndrome coronavirus-2) [1] that represents a serious public health emergency throughout the world
To test the feasibility of our optimized reverse-transcriptase loop-mediated amplification (RT-LAMP) approach, tubes containing conTo test the feasibility of our optimized RT-LAMP approach, tubes containing conventional or optimized RT-LAMP reagents were combined with the target RNA sequence ventional or optimized RT-LAMP reagents were combined with the target RNA sequence
In the optimized RT-LAMP, LAMP, deoxyadenosine triphosphate (dATP) was replaced with dATPαS in the RT-LAMP, whereas the other dNTPs dATP was replaced with dATPαS in the RT-LAMP, whereas the other dNTPs were the were the normal monomers
Summary
COVID-19 (coronavirus disease 2019) is a pandemic disease caused by SARS-CoV-2(severe acute respiratory syndrome coronavirus-2) [1] that represents a serious public health emergency throughout the world. Several different approaches to nucleic acid-based SARS-CoV-2 detection have been published to date [4,5,6,7,8,9,10]. CRISPR/Cas-based approaches have been developed that can detect RNA samples containing over 1 × 104 –1 × 105 copies/mL (SHERLOCK) or 1 × 104 copies/mL (DETECTR) of viral RNA within 1 h. These novel approaches are often relatively complex and/or require access to expensive instrumentation without providing sufficient sensitivity advantages relative to more traditional detection techniques such as RT-LAMP [11]. As such these approaches are generally not convenient or conducive to widespread use
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
