Study: Ninja Omicron: BA.1 subvariant showing a BA.2-like pattern using a variant-specific PCR assay due to a single point mutation downstream the spike 69/70 deletion. Image Credit: Corona Borealis Studio/Shutterstock

SARS-CoV-2 variant-specific polymerase chain reaction assay for SARS-CoV-2 genomic surveillance

In a recent case presentation posted on the Research Square* preprint server researchers reported misclassification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.1 subvariant as the Omicron BA.2 subvariant in an automated variant-specific polymerase chain reaction (vsPCR)- analysis.

Study: Ninja Omicron: BA.1 subvariant showing a BA.2-like pattern using a variant-specific PCR assay due to a single point mutation downstream of the spike 69/70 deletion† Image Credit: Corona Borealis Studio/Shutterstock

Variant tracking is vital for SARS-CoV-2 genomic surveillance. Next-generation sequencing (NGS) is a commonly used technique for identifying variants, it is time consuming and economically unfeasible. The vsPCR assay is a faster and more cost-effective method for detecting variant-determining mutations and relies on amplification (in the case of mutations) or certain peaks in melting temperatures that occur after amplification.

About the case report

In the present case presentation, researchers reported misinterpretation of Omicron BA.1 found as Omicron BA.2 in a vsPCR analysis due to a point mutation.

SARS-CoV-2 ribonucleic acid (RNA) was extracted from patients with coronavirus disease 2019 (COVID-19) for NGS and the vsPCR assays. Bioinformatics analyzes were performed using a custom pipeline and the placement of ultra-fast samples on existing trees UShER) genome for determining SARS-CoV-2 variants.

A discrepancy was found in the results of NGS and vsPCR analyzes in March 2022 for 17 COVID-19 samples from Vigo, Spain. An Omicron BA.1.1.14 cluster showed a melting temperature pattern similar to that of Omicron BA.2 due to the presence of the C21772T point mutation two bases downstream of the deletion of the SARS-CoV-2 spike (S) protein amino acids 69 /70 (referred to as 69/70del).

The 69/70del has been widely used to discriminate between Omicron BA.1 (69/70 deletion positive) and Omicron BA.2 (69/70 deletion negative) by vsPCR and therefore the C21772T mutation may cause misinterpretations of the Omicron BA cause. 1 sub-variant as the Omicron BA.2 sub-variant. More than a thousand sequences of Omicron BA.1 included in the database of the Global All Influenza Data Sharing Initiative (GISAID) carry the C21772T mutation. The way the 69/70 deletion causes S gene target failure (SGTF), new mutations can cause failure in PCR-based analysis.

The team performed multiple alignments and phylogenetic tree analyzes to confirm that the SARS-CoV-2 infected samples were monophyletic, and when matching to the SARS-CoV-2 Wuhan-Hu-1 strain (used as reference), a few alignments misplace the codon 69/70 deletion. Therefore, the mutation was designated A21766T (and not C21772T) in the Nextclade and CoVSpectrum databases.

The 17 COVID-19 samples were subjected to Hain assays and a second vsPCR analysis for retesting, after which the same results were obtained with Omicron BA.2 subvariant interpretation. After contact tracing, 10 sequences related to high school students and four samples were epidemiologically related.

The A67V (C21762T) mutation upstream of the 69/70 deletion is usually present in Omicron BA.1 variants. The authors suggested that the C21772T point mutation prevented the identification of 69/70 codon deletions and that the 69/70 codon deletion causes loss of amino acids valine (V) and histidine (H). Since the adenine (A)-thymine (T)-cytosine (C), ATT and ATA codons all transform into isoleucine (I), the C21772T mutation did not cause substitutions in the amino acid sequence.


Overall, the case findings showed a misclassification of the Omicron BA.1 subvariant as Omicron BA.2 subvariant due to a point mutation that was two nitrogenous bases downstream of the 69/70 deletion in variant-specific PCR analysis. The authors believe that the case report is the first of its kind to report on the C21772T mutation causing negative results in a 69/70 deletion-targeted vsPCR analysis. The report indicates that mutations in the targets of melting curve-based vsPCR assays can lead to misclassification of SARS-CoV-2 variants and therefore confirmation of vsPCR assay results by NGS could improve the accuracy of SARS-CoV-2 genome surveillance .

A few melting curve-based assays developed before the emergence of Omicron and targeting the N501Y mutation of the SARS-CoV-2 S protein yield negative results for samples of Omicron variants, likely due to mutations around the amino acid 501. Recently released Omicron BA.4 and Omicron BA.5 subvariants carry a certain pattern of mutations unexpected by the test software, justifying the need to constantly update variant tracking software.

In addition to the challenges in SARS-CoV-2 genomic surveillance, the A67V mutation allows for discrimination between the Omicron BA.1 subvariant and the Omicron BA.4/5 subvariants; however, the Omicron BA.4 subvariant and the Omicron BA.5 subvariant have similar genetic constitutions at the 69/70del site, and therefore more targets are needed for vsPCR assays to distinguish between Omicron subvariants.

*Important announcement

Research Square publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, should guide clinical practice/health-related behavior or be treated as established information.

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