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

In a current case presentation posted to the Research Square* preprint server, researchers reported on the misclassification of the extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.1 subvariant because the Omicron BA.2 subvariant in an automatic variant-specific polymerase chain response (vsPCR) evaluation.

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
Examine: 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. Picture Credit score: Corona Borealis Studio/Shutterstock

Variant monitoring is significant for SARS-CoV-2 genomic surveillance. Subsequent-generation sequencing (NGS) is a incessantly used method for the identification of variants, it’s time-consuming and never economically viable. The vsPCR assay is a extra speedy and cost-effective technique for detecting variant-defining mutations and depends upon amplification (within the case of mutations) or explicit peaks in melting temperatures that happen after amplification.

In regards to the case report

Within the current case presentation, researchers reported on a misinterpretation of Omicron BA.1 discovered as Omicron BA.2 in a vsPCR evaluation due to a degree mutation.

SARS-CoV-2 ribonucleic acid (RNA) was extracted from coronavirus illness 2019 (COVID-19) sufferers for NGS and the vsPCR assays. Bioinformatics analyses had been carried out utilizing a custom-made pipeline and the Ultrafast Pattern placement on Present tRees (UShER) genome for figuring out SARS-CoV-2 variants.

A discrepancy was discovered within the outcomes 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 sample much like that of Omicron BA.2 as a result of presence of the C21772T level mutation two bases downstream of the deletion of the SARS-CoV-2 spike (S) protein amino acids 69/70 (known as 69/70del).

The 69/70del has been used extensively for differentiating between Omicron BA.1 (69/70 deletion optimistic) and Omicron BA.2 (69/70 deletion damaging) by vsPCR and due to this fact, the C21772T mutation might trigger misinterpretations of the Omicron BA.1 subvariant because the Omicron BA.2 subvariant. Multiple thousand sequences of Omicron BA.1 listed within the international initiative on sharing all influenza knowledge (GISAID) database bear the C21772T mutation. Within the method through which the 69/70 deletion causes S-gene goal failure (SGTF), novel mutations might trigger failure in PCR-based evaluation.

The staff carried out a number of alignments and phylogenetic tree evaluation for confirming that the SARS-CoV-2-infected samples had been monophyletic, and on aligning towards the SARS-CoV-2 Wuhan-Hu-1 pressure (used as reference) just a few alignments misplaced the codon 69/70 deletion. Due to this fact, the mutation was denoted as A21766T (and never C21772T) within the Nextclade and CoVSpectrum databases.

The 17 COVID-19 samples had been subjected to Hain assays and a second vsPCR evaluation for re-testing, after which the identical outcomes with Omicron BA.2 subvariant interpretation had been obtained. After contact tracing, 10 sequences had been discovered to pertain to highschool college students, and 4 samples had been associated epidemiologically.

The mutation A67V (C21762T) upstream of the 69/70 deletion is normally current in Omicron BA.1 variants. The authors urged that the C21772T level mutation prevented 69/70 codon deletion identification and that the 69/70 codon deletion causes lack of amino acids valine (V) and histidine (H). On condition that the adenine (A)-thymine(T)-cytosine (C), ATT, and ATA codons all remodel into isoleucine (I), the C21772T mutation didn’t trigger substitutions within the amino acid sequence.


Total, the case findings confirmed misclassification of the Omicron BA.1 subvariant as Omicron BA.2 subvariant due to some extent mutation which was two nitrogenous bases downstream from the 69/70 deletion in variant-specific PCR evaluation. The authors consider that the case report is the primary of its type to report the C21772T mutation inflicting damaging leads to a 69/70 deletion-targeted vsPCR evaluation. The report signifies that mutations within the targets of melting curve-based vsPCR assays may cause SARS-CoV-2 variant misclassification and due to this fact, affirmation of vsPCR assay outcomes by NGS might improve the SARS-CoV-2 genomic surveillance accuracy.  

Just a few melting curve-based-assays developed earlier than the emergence of Omicron which goal the N501Y mutation of the SARS-CoV-2 S protein yield damaging outcomes for Omicron variant samples, in all probability due to mutations that encompass the amino acid 501. Furthermore, the not too long ago emerged Omicron BA.4 and Omicron BA.5 subvariants bear a specific sample of mutations sudden by the assay software program, warranting the necessity to always replace variant monitoring softwares.

Additional including to the challenges in SARS-CoV-2 genomic surveillance, the A67V mutation permits discrimination between the Omicron BA.1 subvariant and the Omicron BA.4/5 subvariants; nevertheless, the Omicron BA.4 subvariant and the Omicron BA.5 subvariant have comparable genetic constitutions on the 69/70del website, and due to this fact, extra targets are required for vsPCR assays to tell apart between Omicron subvariants.

*Necessary discover

Analysis Sq. publishes preliminary scientific stories that aren’t peer-reviewed and, due to this fact, shouldn’t be thought to be conclusive, information scientific observe/health-related habits, or handled as established info.

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