Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis

Biosens Bioelectron. 2020 Sep 15;164:112316. doi: 10.1016/j.bios.2020.112316. Epub 2020 May 23.

Abstract

Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.

Keywords: COVID-19; CRISPR; Fluorescent detection; Highly sensitive; Molecular diagnosis; SARS-CoV-2.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Base Sequence
  • Betacoronavirus / genetics
  • Betacoronavirus / isolation & purification*
  • Biosensing Techniques / methods
  • Biosensing Techniques / statistics & numerical data
  • COVID-19
  • CRISPR-Cas Systems*
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology
  • Fluorescent Dyes
  • Genes, Viral
  • High-Throughput Nucleotide Sequencing / methods
  • High-Throughput Nucleotide Sequencing / statistics & numerical data
  • Humans
  • Nucleic Acid Amplification Techniques / methods
  • Nucleic Acid Amplification Techniques / statistics & numerical data
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / virology
  • Predictive Value of Tests
  • RNA, Viral / analysis
  • RNA, Viral / genetics
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / statistics & numerical data
  • SARS-CoV-2
  • Sensitivity and Specificity

Substances

  • Fluorescent Dyes
  • RNA, Viral