tetano
Editor, Senior Moderator
Biosens Bioelectron
. 2021 Jan 28;178:113041.
doi: 10.1016/j.bios.2021.113041. Online ahead of print.
Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification
Sheng Ding[SUP] 1 [/SUP], Gangyi Chen[SUP] 2 [/SUP], Yinghua Wei[SUP] 1 [/SUP], Juan Dong[SUP] 3 [/SUP], Feng Du[SUP] 3 [/SUP], Xin Cui[SUP] 3 [/SUP], Xin Huang[SUP] 3 [/SUP], Zhuo Tang[SUP] 4 [/SUP]
Affiliations
Abstract
The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.
Keywords: COVID-19; LAMP; Proofman; SARS-CoV-2; Sequence-specific.
. 2021 Jan 28;178:113041.
doi: 10.1016/j.bios.2021.113041. Online ahead of print.
Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification
Sheng Ding[SUP] 1 [/SUP], Gangyi Chen[SUP] 2 [/SUP], Yinghua Wei[SUP] 1 [/SUP], Juan Dong[SUP] 3 [/SUP], Feng Du[SUP] 3 [/SUP], Xin Cui[SUP] 3 [/SUP], Xin Huang[SUP] 3 [/SUP], Zhuo Tang[SUP] 4 [/SUP]
Affiliations
- PMID: 33545551
- PMCID: PMC7842130
- DOI: 10.1016/j.bios.2021.113041
Abstract
The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.
Keywords: COVID-19; LAMP; Proofman; SARS-CoV-2; Sequence-specific.