实时荧光定量PCR快速鉴别新型冠状病毒主要变异株

Rapid identification of SARS-CoV-2 main variants using real-time quantitative PCR assay

摘要:

为建立一种快速鉴别严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）的5种主要变异株的TaqMan探针实时荧光定量PCR （real-time quantitative PCR，RT-qPCR）体系，基于SARS-CoV-2野生型及变异株alpha （N501Y、HV69-70del）、beta （E484K、K417N）、gamma （K417T、V1176F）、delta （L452R、T478K）和omicron （H655Y、N679K、P681H）序列设计特异性引物、探针，建立和优化一种鉴别新型冠状病毒（SARS-CoV-2）5种主要变异株的TaqMan探针RT-qPCR方法，并进行该方法的特异性、敏感性、鉴别能力评价。该方法可准确区分出SARS-CoV-2野生型和突变型，与其他呼吸道病原体（n=21）无交叉，显示高特异性。该方法最低检测限为2×10²拷贝/mL，操作简单、快速、成本廉价，可用于监测SARS-CoV-2毒株的变异，精准指导疫情识别与防控。

Abstract:

A TaqMan probe real-time quantitative PCR (RT-qPCR) approach was developed for rapid identification of SARS-CoV-2 main variants. Specific primers and probes were designed based on the sequence of the SARS-CoV-2 wild and main variants alpha (N501Y, HV69-70del), beta (E484K, K417N), gamma (K417T, V1176F), delta (L452R, T478K) and omicron (H655Y, N679K, P681H) genes. The specificity, sensitivity and performance of the RT-qPCR assay were tested. The assay can identify SARS-CoV-2 wild type and main variants efficiently, and has no crossover with a panel of respiratory pathogens (n=21), showing high specificity toward SARS-CoV-2 RNA. The assay's sensitivity was determined to be 2×10²copies/mL. In summary, we developed a simple, rapid and cost-effective RT-qPCR assay that enables identification of SARS-CoV-2 main variants. It can be used to monitor the variation of SARS-CoV-2 strain for accurate identification, prevention and control of outbreaks.