FAQ: Do different SARS-CoV-2 variants impact the effectiveness of the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit?

NEB has developed a web tool (Primer Monitor) to continually monitor registered primer sets for overlapping variants in sequences from GISAID. Using this tool, we identified a prominent variant from some countries with a SNP close to the 3’ end of the 2019-nCoV-2_N2 forward primer included in the Luna kit, which detects the Centers for Disease Control and Prevention (CDC) SARS-CoV-2 N1, N2 targets and human RNase P gene with modifications (click for details) (A). To evaluate the impact of this SNP on the N2 target detection, we prepared two N gene RNA fragments containing the wild type and mutant N2 targets, respectively, by in vitro transcription and quantitated them using the SARS-CoV-2 RNA Control 2 from Twist Bioscience. Using the primer/probe set included in the Luna kit, we observed an average 4.2 Cq delay for the mutant N2 target (B). The limit of detection (LOD) for the mutant N2 target was 25 copies/reaction. Though all the reactions containing 10 copies of the mutant RNA generated amplification signal, only 11 of 24 had a Cq ≤ 40, the cut-off for detection (C).