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 genomic monitoring of SARS-CoV-2

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

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

Variant tracking is essential for genomic surveillance of SARS-CoV-2. Next-generation sequencing (NGS) is a frequently used technique for variant identification, it is time consuming and not economically viable. The vsPCR assay is a faster and more economical method of detecting variant-defining mutations and depends on amplification (in the case of mutations) or particular spikes in melting temperatures that occur after amplification.

About the case report

In the present case presentation, researchers reported a 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 vsPCR testing. Bioinformatics analyzes were performed using a custom pipeline and ultra-fast sample placement on existing trees (UShER) to determine SARS-CoV-2 variants.

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

The 69/70del has been widely used to differentiate Omicron BA.1 (positive deletion 69/70) and Omicron BA.2 (negative deletion 69/70) by vsPCR and therefore the C21772T mutation could cause misinterpretations of the Omicron BA. 1 sub-variant like the Omicron BA.2 sub-variant. Over a thousand Omicron BA.1 sequences listed in the Global Initiative on Sharing All Influenza Data (GISAID) database carry the C21772T mutation. Similar to how the 69/70 deletion causes the S gene target (SGTF) to fail, new mutations could 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 aligned against the SARS-CoV-2 strain Wuhan-Hu-1 (used as a reference), a few alignments have misplaced the deletion of codon 69/70. Therefore, the mutation was noted A21766T (not C21772T) in the Nextclade and CoVSpectrum databases.

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

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 the codon 69/70 deletion and that the codon 69/70 deletion resulted in the loss of the amino acids valine (V) and histidine (H). Since the adenine (A)-thymine (T)-cytosine (C), ATT and ATA codons all change to isoleucine (I), the C21772T mutation did not cause substitutions in the amino acid sequence.


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

A few melting curve-based assays developed before the emergence of Omicron that target the SARS-CoV-2 S protein N501Y mutation give negative results for Omicron variant samples, likely due to mutations that surround amino acid 501. Additionally, the recently emerged Omicron BA.4 and Omicron BA.5 subvariants carry a peculiar pattern of mutations that the assay software unexpectedly warrants, justifying the need for continuous updating variant tracking software.

Further adding to the challenges of SARS-CoV-2 genomic surveillance, the A67V mutation allows 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 makeups at the 69/70del site, and therefore more targets are needed for vsPCR assays to distinguish the subvariants Omicron.

*Important Notice

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

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