Journal of Southern Medical University

Journal of Southern Medical University ›› 2023, Vol. 43 ›› Issue (4): 516-526.doi: 10.12122/j.issn.1673-4254.2023.04.03

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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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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