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

• Published in: Biosensors & bioelectronics Vol. 164; p. 112316
• Main Authors: Huang, Zhen, Tian, Di, Liu, Yang, Lin, Zhen, Lyon, Christopher J., Lai, Weihua, Fusco, Dahlene, Drouin, Arnaud, Yin, Xiaoming, Hu, Tony, Ning, Bo
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15-09-2020
• Subjects: Base Sequence; Betacoronavirus - genetics; Betacoronavirus - isolation & purification; Biosensing Techniques - methods; Biosensing Techniques - statistics & numerical data; Coronavirus Infections - diagnosis; Coronavirus Infections - virology; COVID-19; CRISPR; CRISPR-Cas Systems; Fluorescent detection; Fluorescent Dyes; Genes, Viral; High-Throughput Nucleotide Sequencing - methods; High-Throughput Nucleotide Sequencing - statistics & numerical data; Highly sensitive; Humans; Molecular diagnosis; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Amplification Techniques - statistics & numerical data; Pandemics; Pneumonia, Viral - diagnosis; Pneumonia, Viral - virology; Predictive Value of Tests; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - statistics & numerical data; RNA, Viral - analysis; RNA, Viral - genetics; SARS-CoV-2; Sensitivity and Specificity; COVID-19; Fluorescent detection; CRISPR; SARS-CoV-2; Molecular diagnosis; Highly sensitive
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2020.112316

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.
•Integrated CRISPR for COVID-19 clinical diagnosis.•Rapidly identify SARS-CoV-2-specific RNA signatures at an ultra-low concentration.•Detection environment friendly and compatible for high throughput COVID-19 screening in hospital.•Successful development and validation with swab samples from patients.•Improve diagnosis of individuals with suspected COVID-19 infections.