Real-Time RT-PCR for SARS-CoV-2 Variant Detection

Virus suspensions were placed into TRIzol (Invitrogen, Carlsbad, CA) and RNA extracted using the ZR Viral RNA Kit (Zymo Research, Irvine, CA) as per the manufacturer’s protocol. Real time RT-PCR was carried out using the ABI 7900HT Fast Real-Time PCR system (ABI, Carlsbad, CA). Each reaction was performed using the TaqMan RNA-to-C_T 1-Step kit (ABI) as per the manufacturer’s instructions in a 10 μl reaction. The primers and probes were identical to those used in previously published work [6 (link)]. Each probe had a corresponding primer set that was designed to anneal flanking the polymorphic region of each variant. Every sample, run in duplicate, was tested for each variant. Positive and negative controls were run on each plate and all 8 clones were included as controls to ensure no cross-detection of the other clones by an individual probe. Additionally, serial dilutions with titers from 10⁵−10¹ pfu/ml of the individual clones were used to create standard curves.