四重荧光RT-PCR快速鉴别新冠病毒Omicron BA.1、BA.2、BA.4和BA.5变异株

Establishment of quadruple Real-time RT-PCR for the identification of SARS-CoV-2 Omicron BA.1,BA.2,BA.4 and BA.5 variants

目的建立一种四重荧光RT-PCR方法,快速、低成本区分新冠病毒Omicron BA.1、BA.2、BA.4和BA.5变异株。方法比对100个新冠病毒Omicron变异株的基因序列,建立检测N、S、ORF1a和M基因的多重荧光RT-PCR体系,具体包括:针对N基因31/32/33位氨基酸缺损的检测、针对S基因69/70位氨基酸缺损的检测、针对ORF1a基因141/142/143位氨基酸缺损的检测,以及针对M基因D3N氨基酸点突变的检测。用该体系检测80份新冠病毒核酸样本,初判病毒变异株种类。全部样本采用二代测序获得全序列并分型,将初判结果与二代测序的分型结果进行比较,评价该方法的准确性。结果多重荧光RT-PCR检测体系可特异检测新冠病毒,不受其他呼吸道病毒影响。80份新冠病毒核酸样本,用建立的荧光RT-PCR体系进行分型,Omicron变异株49份,其中9份为Omicron BA.1,15份为Omicron BA.2,3份为Omicron BA.4,22份为Omicron BA.5,与二代测序的分型结果完全一致。结论建立的荧光RT-PCR检测体系可准确鉴别Omicron BA.1、BA.2、BA.4和BA.5变异株,为普通实验室提供了快速、简便、准确的新冠病毒变异监测策略。

Objective To rapid and low-cost distinguishing SARS-CoV-2 Omicron BA.1,BA.2,BA.4 and BA.5 variants,we developed a quadruple Real-time RT-PCR method.Methods One hundred sequences of Omicron variants of SARS-CoV-2 strains were compared.The multiplex fluorescence RT-PCR detection system was established to detect N,S,ORF1a and M genes of SARS-CoV-2 Omicron variant,including 31/32/33 aa delete in N gene,69/70 aa delete in S gene,141/142/143 aa delete in ORF1a gene,and D3N aa point mutation in M gene.Using this detection system to test SARS-CoV-2 RNA samples,the viral variants were preliminarily identified.All the samples were sequenced using next-generation sequencing(NGS)to obtain the full sequence and identify variants.The accuracy of this method was evaluated by comparing with NGS.Results The multiplex fluorescence RT-PCR detection system was specific for detecting SARS-CoV-2 and was not affected for other respiratory viruses.80 SARS-CoV-2 RNA samples were identified by the quadruple RT-PCR detection system,there were 49 cases of Omicron variants,including 9 cases of Omicron BA.1,15 cases of Omicron BA.2,3 cases of Omicron BA.4,and 22 cases of Omicron BA.5,which was in perfect accordance with the results of NGS typing.Conclusion The quadruple Real-time RT-PCR detection system could accurately identify Omicron BA.1,BA.2,BA.4 and BA.5 variants.It provided rapid,simple and accurate SARS-CoV-2 variants surveillance strategy for general laboratories.