Coronavirus

PCR Protocol

For qPCR analysis, primers based on nCoV-2019 S gene was designed: RBD-qF1: 5’-  CAATGGTTTAACAGGCACAGG-3’; RBD-qR1: 5’- CTCAAGTGTCTGTGGATCACG-3’. RNA extracted from above used in qPCR by  HiScript® II One Step qRT-PCR SYBR® Green Kit (Vazyme Biotech Co.,Ltd).  onventional PCR test was also performed using the following primer pairs: ND-  CoVs-951F TGTKAGRTTYCCTAAYATTAC; ND-CoVs-1805R  ACATCYTGATANARAACAGC13. The 20 μl qPCR reaction mix contained 10 μl 2×  One Step SYBR Green Mix, 1 μl One Step SYBR Green Enzyme Mix, 0.4 μl 50 ×   ROX Reference Dye 1, 0.4 μl of each primer (10 uM) and 2 μl template RNA.  Amplification was performed as follows: 50 0C for 3 min, 95 0C for 30 s followed by  40 cycles consisting of 95 0C for 10 s, 60 0C for 30 s, and a default melting curve step in an ABI 7700 machine.