Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay

Angew Chem Int Ed Engl. 2021 Mar 1;60(10):5307-5315. doi: 10.1002/anie.202014506. Epub 2021 Jan 26.

Abstract

Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.

Keywords: RNA; SARS-CoV-2; genes; polymerases; viruses.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • COVID-19 / diagnosis*
  • COVID-19 / virology
  • CRISPR-Cas Systems*
  • Genes, Viral*
  • Humans
  • Nasopharynx / virology
  • RNA, Viral
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • SARS-CoV-2 / genetics
  • SARS-CoV-2 / isolation & purification*
  • Sensitivity and Specificity

Substances

  • RNA, Viral