Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription-free exponential amplification reaction, RTF-EXPAR

被引:59
作者
Carter, Jake G. [1 ,2 ]
Iturbe, Lorea Orueta [3 ]
Duprey, Jean-Louis H. A. [3 ]
Carter, Ian R. [4 ]
Southern, Craig D. [4 ]
Rana, Marium [1 ,2 ]
Whalley, Celina M. [5 ]
Bosworth, Andrew [5 ,6 ]
Beggs, Andrew D. [5 ]
Hicks, Matthew R. [3 ]
Tucker, James H. R. [1 ]
Dafforn, Timothy R. [2 ]
机构
[1] Univ Birmingham, Sch Chem, Birmingham B15 2TT, W Midlands, England
[2] Univ Birmingham, Sch Biosci, Birmingham B15 2TT, W Midlands, England
[3] Linear Diagnost Ltd, Birmingham B15 2SQ, W Midlands, England
[4] C2JF Solut LLP, Liverpool L24 9LG, Merseyside, England
[5] Univ Birmingham, Inst Canc & Genom, Birmingham B15 2TT, W Midlands, England
[6] Univ Hosp Birmingham NHS Fdn Trust, Clin Lab Serv, Clin Virol, Birmingham B15 2GW, W Midlands, England
关键词
RNA detection  COVID-19 assay  nucleic acids  EXPAR  isothermal amplification; ISOTHERMAL AMPLIFICATION; SYSTEM;
D O I
10.1073/pnas.2100347118
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription-free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct threeway comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.
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页数:6
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