SARS-CoV-2 Major Variant Strains Multiplex RT-qPCR Screening Kit

Coronaviruses are a family of large RNA viruses with size ranging from 26 to 32 kb. These viruses are zoonotic and in humans can cause respiratory infections. As the coronavirus is an RNA virus, it has a relatively high mutation rate resulting in rapid evolution. In December 2019, a new deadly coronavirus known as SARS-CoV-2, which has a high sequence similarity to SARS-CoV, was identified as the cause of the Covid-19 outbreak. Since then, numerous variants of SARS-CoV-2 were reported around the world. Among them, several major variant strains, including B.1.1.7 (also known as 501Y.V1), B.1.351 (also known as 501Y.V2), P.1, and P.2, have gained enormous attention. These variant strains show increased transmissibility and have become major strains spreading globally.
Several mutations identified in these strains are thought responsible for the higher infection rates and immune evasion. These mutations include spike-HV 69-70 deletion, the spike-N501Y mutation, and the spike-E484K mutation. The spike-HV 69-70 deletion mutation may promote viral immune escape and is considered the hallmark of the B.1.1.7 lineage, as it is not present in other major circulating SARS-CoV-2 strains including 501Y.V2, P.1, and P.2. The spike-N501Y mutation may contribute to the higher viral binding affinity to the ACE2 receptor on the human cell membrane, and is present in some major circulating SARS-CoV-2 strains including B.1.1.7, 501Y.V2, and P.1. The spike-E484K mutation may contribute to the immune evasion of SARS-CoV-2 and dampen current vaccine efficacy. This mutation is present in some major circulating SARS-CoV-2 strains including 501Y.V2, P.1 and P.2.

• Variant: B.1.1.7 - Mutations: HV69-70 deletion, N501Y
• Variant: B.1.351 - Mutations: N501Y, E484K
• Variant: P.1 - Mutations: N501Y, E484K
• Variant: P.2 - Mutations: E484K

ScienCell's SARS-CoV-2 Major Variant Strains Multiplex RT-qPCR Screening Kit (SCVVS) is designed to screen for the SARS-CoV-2 variant strain presence by detecting these major mutations. Four multiplex primer/probe set components (Cat #7148-REF, #7118-H69V70, #7128-N501Y, and #7138-E484K) are included in the kit. The reference primer/probe set component (Cat #7148-REF) contains 3 primer/probe sets, N1-FAM, N2-FAM, and RP-HEX (Table 2). Among them, N1-FAM and N2-FAM target two regions on the coronavirus SARS-CoV-2 nucleocapsid (N) gene, and RP-HEX targets the exon 1 of human RPP30 gene and serves as a control to assess specimen quality. The HV 69-70 primer/probe set component (Cat #7118-H69V70) contains 2 primer/probe sets, H69V70-Del-FAM and H69V70-Present-HEX (Table 3), which target the coronavirus SARS-CoV-2 spike (S) gene with HV 69-70 deleted and present, respectively. The N501Y primer/probe set component (Cat #7128-N501Y) contains 2 primer/probe sets, Y501-FAM and N501-HEX (Table 4), which target the coronavirus SARS-CoV-2 spike (S) gene with a tyrosine (Y) and an asparagine (N) at position 501, respectively. The E484K primer/probe set component (Cat #7138-E484K) contains 2 primer/probe sets, K484-FAM and E484-HEX (Table 5), which target the coronavirus SARS-CoV-2 spike (S) gene with a lysine (K) and a glutamic acid (E) at position 484, respectively. For more efficient screening, if the expected mutation rate is low, a pool of up to 10 RNA samples can be used as the template for one qPCR reaction. If results for H69V70-Del-FAM, Y501-FAM, or K484-FAM are negative, then all pooled samples do not contain the spike-HV 69-70 deletion mutation, N501Y, or E484K mutation, respectively. For H69V70-Del-FAM, Y501-FAM, or K484-FAM positive pooled samples, the pooled samples should be tested individually to identify the mutated one(s).

Primer/probe set list of the reference primer/probe set component (Cat #7148-REF)

• Set: N1-FAM, Target: SARS-CoV-2 nucleocapsid (N) gene, region 1, Dye: FAM
• Set: N2-FAM, Target: SARS-CoV-2 nucleocapsid (N) gene, region 2, Dye: FAM
• Set: RP-HEX, Target: Human RPP30 gene, Dye: HEX

Primer/probe set list of the HV 69-70 primer/probe set component (Cat #7118-H69V70)

• Set: H69V70-Del-FAM, Target: SARS-CoV-2 spike (S) gene, HV 69-70 deleted, Dye: FAM
• Set: H69V70-Present-HEX, Target: SARS-CoV-2 spike (S) gene, HV 69-70 present, Dye: HEX

Primer/probe set list of the N501Y primer/probe set component (Cat #7128-N501Y)

• Set: Y501-FAM, Target: SARS-CoV-2 spike (S) gene, N501Y mutation, Dye: FAM
• Set: N501-HEX, Target: SARS-CoV-2 spike (S) gene, N501 not mutated, Dye: HEX

Primer/probe set list of the E484K primer/probe set component (Cat #7138-E484K)

• Set: K484-FAM, Target: SARS-CoV-2 spike (S) gene, E484K mutation, Dye: FAM
• Set: E484-HEX, Target: SARS-CoV-2 spike (S) gene, E484 not mutated, Dye: HEX/ul>In addition, ScienCell One-Step TaqProbe RT-qPCR master mix (Cat #MB802a), a non-infectious positive control (Cat #7148-Pos), and nuclease-free water (Cat #7148-H2O) are included in the kit. The positive control (Cat #7148-Pos) consists of non-infectious viral RNA fragments of the original strain, the B.1.1.7 lineage, the B.1.351 lineage, the P.1 lineage, and the P.2 lineage of SARS-CoV-2 spiked into human small airway epithelial cells.

ScienCell's SARS-CoV-2 Major Variant Strains Multiplex RT-qPCR Screening Kit (SCVVS) is designed to screen for the SARS-CoV-2 variant strain presence by detecting these major mutations. For Research Use Only. Not for use in diagnostic procedures.

Kit Components:

• #MB802a - One-Step TaqProbe RT-qPCR master mix, 4x - 2 x 1.5 mL - Storage: -20°C
• #7148-REF - Reference multiplex primer/probe sets, in solution - 600 uL - Storage: -20°C
• #7118-H69V70 - HV 69-70 multiplex primer/probe sets, in solution - 600 uL - Storage: -20°C
• #7128-N501Y - N501Y multiplex primer/probe sets, in solution - 600 uL - Storage: -20°C
• #7138-E484K - E484K multiplex primer/probe sets, in solution - 600 uL - Storage: -20°C
• #7148-H2O - Nuclease-free H2O - 4 mL - Storage: 4°C
• #7148-Pos - Positive control (non-infectious; RNA: 500 - 1000 copies/uL, cells: 200 - 300 counts/uL) - 200 uL - Storage: -80°C