南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (4): 516-526.doi: 10.12122/j.issn.1673-4254.2023.04.03

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RT-PCR/CRISPR-Cas12a快速检测和区分SARS-CoV-2Omicron BA.4/5变异株

马玉楠，邹丽容，梁源浩，刘泉汛，孙 倩，庞玉莲，林洪青，邓小玲，唐时幸   

  1. 南方医科大学公共卫生学院流行病学系，广东 广州 510515；广东省疾病预防控制中心病原微生物研究所，中国医学科学院广东省新发传染病防治工作站，广东 广州 511430；南方医科大学南方医院感染科，广东 广州 510515
  • 出版日期:2023-04-20 发布日期:2023-05-16

Rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants using a RT-PCR and CRISPR-Cas12a-based assay

MA Yu'nan, ZOU Lirong, LIANG Yuanhao, LIU Quanxun, SUN Qian, PANG Yulian, LIN Hongqing, DENG Xiaoling, TANG Shixing   

  1. Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou 510515, China; Institute of Pathogenic Microbiology, Guangdong Provincial Center for Disease Control and Prevention, Guangdong Workstation for Emerging Infectious Disease Control and Prevention, Chinese Academy of Medical Sciences, Guangzhou 511430, China; Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
  • Online:2023-04-20 Published:2023-05-16

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摘要： 目的 建立一种基于CRISPPR-Cas12a基因编辑技术对新型冠状病毒（SARS-CoV-2）奥密克戎BA.4/5变异株快速检测与鉴别的方法。方法 结合逆转录聚合酶链式反应（RT-PCR）与 CRISPR 基因编辑技术，基于次优原间隔区相邻基序（suboptimal-PAM）设计奥密克戎BA.4/5变异株特异性的CRISPR RNA（crRNA），从而建立RT-PCR/CRISPR-Cas12a快速检测新冠病毒BA.4/5变异株的方法。本研究检测了43例新冠病毒阳性的临床样本（包括新冠病毒野生株以及变异株Alpha、Beta、Delta、奥密克戎BA.1和BA.4/5）以及20例新冠病毒阴性的临床样本（包括11种常见呼吸道病原体），并以测序结果为金标准计算PCR/CRISPR-Cas12a检测方法的ROC 曲线下面积（AUC）以及灵敏度（SEN）、特异性（SPE）、一致性（Kappa）评价检测方法的性能。结果 本研究筛选到两条特异性crRNA-1和crRNA-2，所建立的方法可在30 min内快速特异地检测出SARS-CoV-2奥密克戎BA.4/5变异株，最低检测限为10 copies/μL。此外，在检测感染11种常见呼吸道病原体的临床样本时均未观察到非特异性交叉反应。基于crRNA-1和crRNA-2的检测结果的灵敏度分别为97.83%、100%；特异性均为100%；ROC 曲线下面积分别为 0.9989 和 1.0；与 Sanger 测序方法的一致性分别为 92.83%、96.41%。结论 本研究采用 CRISPPR-Cas12a基因编辑技术与逆转录聚合酶链式反应相结合，成功建立了一种快速检测与鉴别SARS-CoV-2奥密克戎BA.4/5变异株的新方法。其特异性强、灵敏度高、稳定性好，可用于新冠病毒奥密克戎BA.4/5变异株的批量检测与分型，可用于常规监测和跟踪SARS-CoV-2变异的传播。

关键词: 聚合酶链式反应；CRISPR基因编辑；新冠核酸检测；奥密克戎；BA.4/5变异株

Abstract: Objective To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology. Methods We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants. The performance of this RT-PCR/CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild- type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated. Results This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively. Conclusion By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.

Key words: polymerase chain reaction; CRISPR gene editing; SARS-CoV-2 nucleic acid test; Omicron BA.4/5 variants