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

In a recent case presentation posted to the Research Square* preprint server, researchers reported on the 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 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 frequently used technique for the identification of variants, it is time-consuming and not economically viable. The vsPCR assay is a more rapid and cost-effective method for detecting variant-defining mutations and depends on amplification (in the case of mutations) or particular peaks in melting temperatures that occur after amplification.

About the case report

In the present case presentation, researchers reported on 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 coronavirus disease 2019 (COVID-19) patients for NGS and the vsPCR assays. Bioinformatics analyses were performed using a customized pipeline and the Ultrafast Sample placement on Existing tRees (UShER) genome for determining SARS-CoV-2 variants.

A discrepancy was found in the results of NGS and vsPCR analyses in March 2022 for 17 COVID-19 samples from Vigo, Spain. An Omicron BA.1.1.14 cluster demonstrated 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 used widely for differentiating between Omicron BA.1 (69/70 deletion positive) and Omicron BA.2 (69/70 deletion negative) by vsPCR and therefore, the C21772T mutation could cause misinterpretations of the Omicron BA.1 subvariant as the Omicron BA.2 subvariant. More than one thousand sequences of Omicron BA.1 listed in the global initiative on sharing all influenza data (GISAID) database bear the C21772T mutation. In the manner in which the 69/70 deletion causes S-gene target failure (SGTF), novel mutations could cause failure in PCR-based analysis.

The team performed multiple alignments and phylogenetic tree analysis for confirming that the SARS-CoV-2-infected samples were monophyletic, and on aligning against the SARS-CoV-2 Wuhan-Hu-1 strain (used as reference) a few alignments misplaced the codon 69/70 deletion. Therefore, the mutation was denoted as 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 re-testing, after which the same results with Omicron BA.2 subvariant interpretation were obtained. After contact tracing, 10 sequences were found to pertain to high school students, and four samples were related epidemiologically.

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


Overall, the case findings showed misclassification of the Omicron BA.1 subvariant as Omicron BA.2 subvariant because of a point mutation which was two nitrogenous bases downstream from the 69/70 deletion in variant-specific PCR analysis. The authors believe that the case report is the first of its kind to report 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 cause SARS-CoV-2 variant misclassification and therefore, confirmation of vsPCR assay results by NGS could enhance the SARS-CoV-2 genomic surveillance accuracy.  

A few melting curve-based-assays developed before the emergence of Omicron which target the N501Y mutation of the SARS-CoV-2 S protein yield negative results for Omicron variant samples, probably because of mutations that surround the amino acid 501. Moreover, the recently emerged Omicron BA.4 and Omicron BA.5 subvariants bear a particular pattern of mutations unexpected by the assay software, warranting the need to constantly update variant tracking softwares.

Further adding to the challenges in SARS-CoV-2 genomic surveillance, the A67V mutation enables 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 required for vsPCR assays to distinguish between Omicron subvariants.

*Important notice

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

Journal reference:
  • Carlos Daviña-Nuñez, Sonia Pérez-Castro, Lucía Martínez-Lamas et al. (2022). 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. Research Squaredoi: https://doi.org/10.21203/rs.3.rs-1730154/v1 https://www.researchsquare.com/article/rs-1730154/v1

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: Adenine, Amino Acid, Assay, Bases, Bioinformatics, Codon, Coronavirus, Coronavirus Disease COVID-19, covid-19, Cytosine, Gene, Genetic, Genome, Genomic, Histidine, Influenza, Isoleucine, Mutation, Omicron, Point mutation, Polymerase, Polymerase Chain Reaction, Protein, Research, Respiratory, Ribonucleic Acid, RNA, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, students, Syndrome, Thymine, Valine

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Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.

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  • Posted on June 17, 2022