Variant detection by real-time PCR
ENDO
Allplex SARS-CoV-2 Variants I Assay from Seegene Inc was used according to the manufacturer protocol to perform rRT-PCR. Briefly, Extracted RNA (5 µl) was transferred to 96 well PCR plate containing 15 µll of the master mix. Plates were then spun down at 2500 rpm for 5 s and analyzed on a CFX96 Touch Real-Time PCR from BioRad. Reverse Transcription reaction 1 cycle: 50 °C/20 min – 95 °C/15 min. PCR reaction 45 cycles: 94 °C/15 s – 58 °C/30 sec. Gene amplifications were analyzed by FAM (E484K mutation on S-Gene), HEX (RdRP), Cal Red 610 (N501Y mutation on S-Gene), Quasar 705 (69-70del on S-Gene), and Quasar 670 (Human Endo Internal control) fluorophores. Results were compiled and analyzed using the 2019-nCoV viewer from Seegene Inc according to the manufacturer’s instructions.