Several variants of SARS-CoV-2 have emerged bringing challenges to diagnostic tests and pandemic eradication efforts. Of particular significance are variants B.1.1.7 (also known as Alpha, first identifed in the United Kingdom), B.1.351 (also known as Beta, first identified in South Africa), B.1.1.28 (also known as P.1 or Gamma, first identified in travellers from Brazil who arrived in Japan) and B.1.617 (including the Kappa and Delta variants, first identified in India). The ability to quickly and reliably identify SARS-CoV-2 mutations will enhance the ability to gather crucial public health information regarding transmissibility kinetics of new variants, and the efficacy of vaccines and therapeutics.

SARS-CoV-2 Variant ValuPanel assays consist of separately delivered probes and primers that are designed for qualitative detection of specific SARS-CoV-2 mutations by RT-PCR-based genotyping. Each SARS-CoV-2 Variant ValuPanel assay consists of 2 BHQplus™ probes and 2 primers for amplification and simultaneous discrimination between a single mutation and the wild-type SARS-CoV-2 sequence.

SARS-CoV-2 Variant ValuPanel assays are for research use only, they are not intended for SARS-CoV-2 diagnosis. The assays are intended for secondary, informational tests only, designed for variant screening in respiratory tract samples that have previously tested positive for SARS-CoV-2 by diagnostic RT-PCR.

Each panel contains the following:

• Forward and reverse primers that amplify the SARS-CoV-2 target sequence. One primer also initiates reverse transcription of the target sequence.
• BHQplus™ probes that specifically detect the mutation site
  • A mutation-specific probe labeled with CAL Fluor Orange 560
  • A reference/wild type-specific probe labeled with FAM

Probes and primers are supplied in dried format, at 5 nmol of each probe and 20 nmol of each primer. Probes and primers are delivered in separate tubes, they are not premixed.

Each kit can perform at least 1000 reactions, based on 20 uL reactions containing 200 nM/probe and 900 nM/primer. Use our Reaction Estimator Tool to calculate the number of reactions achievable when using other reaction volumes and oligo concentrations.

SARS-CoV-2 Variant ValuPanel assays are designed to be compatible with all qPCR instruments that are capable of detecting FAM and CAL Fluor Orange 560 (VIC/HEX/JOE channel).

Materials not included in the kit:
PCR master mix, ROX reference dye, and positive controls are not included. We recommend AccuPlex SARS-CoV-2 variant positive controls from LGC SeraCare Life Sciences. See the manual for additional positive control template options.

SARS-CoV-2 Variant ValuPanel assays

Amino acid mutations present in SARS-CoV-2 variants

Important considerations for detecting the Omicron variant

The Omicron sequence contains proximal mutations that have varying effects on the performance of the SARS-CoV-2 Variant ValuPanel assays.

• The Del H69-V70 assay drops out in Omicron samples. This is likely due to the proximal mutation (A67V) which is present in the binding sequence for the deletion/mutation-specific probe. While this assay can’t be used to specially detect the H69-V70 deletion in Omicron samples, it can be used for S gene dropout assays, also called S-gene target failure (SGTF).
• The E484K assay drops out in Omicron samples due to several proximal mutations in the assay.
• The N501Y assay tolerates the proximal mutations in Omicron samples. Assays show reduced fluorescence on Omicron samples (as compared to N501Y detection on the Alpha variant), but it still establishes a distinct scorable cluster along the CAL Fluor™ Orange 560 axis for Omicron samples.
• The P681H assay tolerates the proximal mutations in the Omicron lineage. The assay shows reduced fluorescence on Omicron samples (as compared to Alpha), but still establishes a distinct scorable cluster. Please note P681H can generate a “heterozygote” cluster due to the P681R mutation present in some variants including Delta and Kappa.
• The K417N assay performs well on Omicron samples as there are no proximal mutations in this region. For accurate genotyping of tri-allelic mutations we recommend that both assays at each position are used, such as assays for both K417T and K417N. Please see the manual for more information.

Don’t see the assays that you want? Our Assay Design and Development team is here to help; and can provide custom assay designs for any SARS-CoV-2 mutation. Contact us at ADD@LGCgroup.com

*The Omicron sequence contains proximal mutations at these sites, which may affect the performance of the SARS-CoV-2 Variant ValuPanel assays. Please see above for important considerations for detecting the Omicron variant.

Analysis of tri-allelic codons:
Mutations occurring at amino acid positions 417, 484 and 681 of the spike protein are attractive targets for genotyping analysis, but additional analysis may be required due to multiple mutations arising within these codons. For accurate genotyping at these positions we recommend that both assays at each position are used. For example, we recommend running both K417T and K417N assays, or both E484K and E484Q, or both P681H and P681R. Concordant results from these companion assays can compensate for the challenges of genotyping tri-allelic codons. Please see the manual for more information.

P681H assay:
Please note: We replaced the first version of the P681H assay (Cat. No. SCV-P681H-1000) with SARS-CoV-2 Variant ValuPanel [P681H] (Version 2), which was designed to avoid the potential proximal mutation at Q677 and demonstrates improved cluster separation in endpoint genotyping analysis

*For Research Use Only. Not for use in diagnostic procedures.

Certificates of Analysis are available upon request for each oligonucleotide. Please contact techsupport@lgcgroup.com to obtain your copies.