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    基于逆转录微滴式数字PCR的污水中SARS-CoV-2变异株检测方法的建立

    Establishment of a detection method for SARS-CoV-2 variants in wasterwater based on reverse transcription droplet digital PCR

      摘要
    • 摘要:
      目的 旨在建立检测和绝对定量新型冠状病毒(SARS-CoV-2)变异株的方法。
      方法 以SARS-CoV-2变异株BF.7.14为例, 针对变异株突变位点保守区域分别设计特异引物和探针, 建立检测SARS-CoV-2的逆转录微滴式数字PCR(RT-ddPCR)方法, 评价其特异性、灵敏性和适用性等。
      结果 方法在引物探针浓度分别为900、250 nmol/L, 退火温度为52.5 ℃时优化结果最佳; 建立的方法能够准确检出目标SARS-CoV-2变异株的同时, 降低其他变异株的干扰; 采用该建立的方法检测SARS-CoV-2变异株的定量限为0.28拷贝/μL; 对18个实际样本分别进行RT-ddPCR和RT-qPCR检测, 检出率均为100%(18/18), RT-ddPCR检出浓度范围为16~7 648拷贝/mL, RT-qPCR检出浓度范围为14~4 875拷贝/mL。
      结论 RT-ddPCR方法能够很好地检测污水中SARS-CoV-2变异株。

       

      Abstract:
      Objective To develop a method for detection and absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants.
      Methods Taking SARS-CoV-2 BF.7.14 as an example, specific primers and probes were designed for the conserved region of the mutation site of the strain to establish a reverse transcription droplet digital PCR (RT-ddPCR) method. Its specificity, sensitivity, and applicability were evaluated.
      Results The optimal conditions were: primer concentration, 900 nmol/L; probe concentration, 250 nmol/L; and annealing temperature, 52.5 ℃. By this method, the target SARS-CoV-2 variant could be accurately detected, and interference from other variants was reduced at the same time. The limit of detection for the SARS-CoV-2 variant using this method was 0.28 copies/μL. For 18 actual samples, both RT-ddPCR and RT-qPCR showed a detection rate of 100% (18/18), and the concentration range was 16-7 648 copies/mL with RT-ddPCR and 14-4 875 copies/mL with RT-qPCR.
      Conclusion RT-ddPCR is an effective method for the detection of SARS-CoV-2 variants in wastewater.

       

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