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

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

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CRISPRRNA(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30min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10copies/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。

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 rapidand 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.