Koshikawa Takuro, Miyoshi Hiroshi
Department of Microbiology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, 216-8511, Japan.
Biochem Biophys Rep. 2022 Sep;31:101306. doi: 10.1016/j.bbrep.2022.101306. Epub 2022 Jul 1.
High-resolution melting (HRM) analysis was conducted to discriminate between SARS-CoV-2 Omicron variant BA.1 (B.1.1.529.1) and subvariant BA.2 (B.1.1.529.2). We performed two-step PCR consisting of the first PCR and the second nested PCR to prepare the amplicon for HRM analysis, which detected G339D, N440K, G446S and D796Y variations in the SARS-CoV-2 spike protein. The melting temperatures (Tms) of the amplicons from the cDNA of the Omicron variant BA.1 and subvariant BA.2 receptor binding domain (RBD) in spike protein were the same: 75.2 °C (G339D variation) and 73.4 °C (D796Y variation). These Tms were distinct from those of SARS-CoV-2 isolate Wuhan-Hu-1, and were specific to the Omicron variant. In HRM analyses that detected the N440K and G446S variations, the Tms of amplicons from the cDNA of the Omicron variant BA.1 and subvariant BA.2 RBDs were 73.0 °C (N440K and G446S variations) and 73.5 °C (G446S variation). This difference indicates that the SARS-CoV-2 Omicron variants BA.1 and BA.2 can be clearly discriminated. Our study demonstrates the usefulness of HRM analysis after two-step PCR for the discrimination of SARS-CoV-2 variants.
进行了高分辨率熔解（HRM）分析，以区分严重急性呼吸综合征冠状病毒2（SARS-CoV-2）奥密克戎变异株BA.1（B.1.1.529.1）和亚变异株BA.2（B.1.1.529.2）。我们进行了两步PCR，包括第一次PCR和第二次巢式PCR，以制备用于HRM分析的扩增子，该扩增子检测了SARS-CoV-2刺突蛋白中的G339D、N440K、G446S和D796Y变异。来自奥密克戎变异株BA.1和亚变异株BA.2刺突蛋白中受体结合域（RBD）的cDNA的扩增子的熔解温度（Tm）相同：75.2℃（G339D变异）和73.4℃（D796Y变异）。这些Tm与SARS-CoV-2分离株武汉-胡-1的不同，并且是奥密克戎变异株特有的。在检测N440K和G446S变异的HRM分析中，来自奥密克戎变异株BA.1和亚变异株BA.2 RBD的cDNA的扩增子的Tm分别为73.0℃（N440K和G446S变异）和73.5℃（G446S变异）。这种差异表明可以清楚地区分SARS-CoV-2奥密克戎变异株BA.1和BA.2。我们的研究证明了两步PCR后进行HRM分析对于区分SARS-CoV-2变异株的有用性。
