SARS-CoV-2 Quantitative RT-PCR Protocol

Reference RT-PCR examinations were performed with purified samples using a method developed by the National Institute of Infectious Diseases (NIID), Japan, for SARS-CoV-2 [17 (link), 18 (link)], which was used Briefly, 5 μL of the extracted RNA was used for one-step quantitative RT-PCR with the THUNDERBIRD^® Probe One-step qRT-PCR kit (TOYOBO Co., Ltd.) and the LightCycler^® 96 Real-time PCR System (Roche Diagnostics KK, Basel, Switzerland). A duplicate analysis for N2 genes was performed for the evaluation of SARS-CoV-2. EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and sterile purified water (Merck & Co., Inc., Kenilworth, NJ, USA) were used as positive and negative controls, respectively. The calibration curves were generated with 5, 50 and 500 copies/reaction of EDX SARS-CoV-2 Standard.