Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2

Yu Jin Jung; Gun-Soo Park; Jun Hye Moon; Keunbon Ku; Seung-Hwa Beak; Seil Kim; Edmond Changkyun Park; Daeui Park; Jong-Hwan Lee; Cheol Woo Byeon; Joong Jin Lee; Jin-Soo Maeng; Seong Jun Kim; Seung Il Kim; Bum-Tae Kim; Min Jun Lee; Hong Gi Kim

Extracted nucleic acid samples were tested for comparative analysis of SARS-CoV-2 by qRT-PCR. The Orf1 and N region of SARS-CoV-2 were used as the target sequences for SARS-CoV-2 specific gene. Briefly, 10 μL of purified viral RNA was amplified in a 20 μL reaction solution containing 1X 1 step RT-PCR mix (WELLS BIO INC., South Korea), and 300 nM of primers and probes for the target detection. The qRT-PCR was performed with a CFX 96 touch real-time PCR detection system (Bio-rad, Hercules, CA, USA). The qRT-PCR conditions applied in this study were programmed as follows: UNG incubation, RT incubation, and enzyme activation were serially performed at 25 °C for 2 minutes, at 55 °C for 10 minutes, at 94 °C for 3 minutes respectively. Thermal cycling was then performed at 94 °C for 15 seconds (denaturation), and at 60 °C for 30 seconds (annealing and amplification) for forty-five cycles.