Highly Sensitive Detection Method for HV69-70del in SARS-CoV-2 Alpha and Omicron Variants Based on CRISPR/Cas13a

Frontiers in Bioengineering and Biotechnology

Xueshan Xia¹  Mengwei Niu¹  Lan Yang²  Hao Li²  Fan Li²  Xue Dong²  Yao Han²  Youcui Zhang²  Qiang Hu²  Yansong Sun² 

[1] Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China;State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China;

关键词: variants;    SARS-CoV-2;    CRISPR/Cas13a;    nucleic acid detection;    lateral flow strip;   

DOI  :  10.3389/fbioe.2022.831332

来源: DOAJ

【 摘 要 】

As SARS-CoV-2 variants continue to evolve, identifying variants with adaptive diagnostic tool is critical to containing the ongoing COVID-19 pandemic. Herein, we establish a highly sensitive and portable on-site detection method for the HV69-70del which exist in SARS-CoV-2 Alpha and Omicron variants using a PCR-based CRISPR/Cas13a detection system (PCR-CRISPR). The specific crRNA (CRISPR RNA) targeting the HV69-70del is screened using the fluorescence-based CRISPR assay, and the sensitivity and specificity of this method are evaluated using diluted nucleic acids of SARS-CoV-2 variants and other pathogens. The results show that the PCR-CRISPR detection method can detect 1 copies/μL SARS-CoV-2 HV69-70del mutant RNA and identify 0.1% of mutant RNA in mixed samples, which is more sensitive than the RT-qPCR based commercial SARS-CoV-2 variants detection kits and sanger sequencing. And it has no cross reactivity with ten other pathogens nucleic acids. Additionally, by combined with our previously developed ERASE (Easy-Readout and Sensitive Enhanced) lateral flow strip suitable for CRISPR detection, we provide a novel diagnosis tool to identify SARS-CoV-2 variants in primary and resource-limited medical institutions without professional and expensive fluorescent detector.

【 授权许可】

Unknown