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 variant) Omicron BA.1 subvariant as the Omicron BA.2 subvariant in an automated variant-specific polymerase chain reaction (vsPCR) analysis.

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

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.

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.

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