Niu Mengwei, Han Yao, Dong Xue, Yang Lan, Li Fan, Zhang Youcui, Hu Qiang, Xia Xueshan, Li Hao, Sun Yansong
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
Front Bioeng Biotechnol. 2022 Apr 12;10:831332. doi: 10.3389/fbioe.2022.831332. eCollection 2022.
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.
随着严重急性呼吸综合征冠状病毒2（SARS-CoV-2）变体不断进化，使用适应性诊断工具识别变体对于控制当前的冠状病毒病（COVID-19）大流行至关重要。在此，我们使用基于聚合酶链反应（PCR）的CRISPR/Cas13a检测系统（PCR-CRISPR），建立了一种针对存在于SARS-CoV-2 Alpha和奥密克戎变体中的HV69-70del的高灵敏度便携式现场检测方法。使用基于荧光的CRISPR检测筛选靶向HV69-70del的特异性CRISPR RNA（crRNA），并使用SARS-CoV-2变体和其他病原体的稀释核酸评估该方法的灵敏度和特异性。结果表明，PCR-CRISPR检测方法可检测到1拷贝/μL的SARS-CoV-2 HV69-70del突变体RNA，并能识别混合样本中0.1%的突变体RNA，比基于逆转录定量聚合酶链反应（RT-qPCR）的商用SARS-CoV-2变体检测试剂盒和桑格测序更灵敏。并且它与其他十种病原体核酸无交叉反应。此外，通过与我们之前开发的适用于CRISPR检测的易读且灵敏增强（ERASE）侧向流动试纸条相结合，我们提供了一种新型诊断工具，可在没有专业且昂贵的荧光检测仪的基层和资源有限的医疗机构中识别SARS-CoV-2变体。
