SARS-CoV-2 mutant-specific polymerase chain reaction assay for SARS-CoV-2 genome surveillance

In recent cases, the presentation was posted to Research Square* Preprint server, researchers have found that in automated variant-specific polymerase chain reaction (vsPCR) analysis, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Omicron BA.1 subvariant Omicron BA.2 Reported misclassification as a subvariant.

Variant tracking is essential for SARS-CoV-2 genome monitoring. Next Generation Sequencing (NGS) is a frequently used technique for identifying variants, which is time consuming and not economically feasible. The vsPCR assay is a faster and more cost-effective way to detect mutations that define mutations, depending on the specific peak of amplification (in the case of mutations) or the melting temperature that occurs after amplification.

About case reports

In this case presentation, researchers reported a misunderstanding of Omicron BA.1 that was discovered as Omicron BA.2 in a point mutation vs PCR analysis.

SARS-CoV-2 ribonucleic acid (RNA) was extracted from patients with coronavirus disease 2019 (COVID-19) for NGS and vsPCR assays.Bioinformatics analysis was performed using a customized pipeline and ultrafast sample placement on existing trees. ((((UShER) Genome for determining SARS-CoV-2 mutants.

Inconsistencies were found in the results of the March 2022 NGS and vs PCR analysis for 17 COVID-19 samples from Vigo, Spain. The Omicron BA.1.1.14 cluster showed a melting temperature pattern similar to Omicron BA.2 due to the presence of a C21772T point mutation 2 bases downstream of the SARS-CoV-2 spike (S) protein amino acid deletion. rice field. 69/70 (called 69 / 70del).

The C21772T mutation is of Omicron BA because 69/70 del is widely used to distinguish between Omicron BA.1 (69/70 deletion positive) and Omicron BA.2 (69/70 deletion negative) by vsPCR. It can be misleading. One subvariant as the Omicron BA.2 subvariant. Over 1000 Omicron BA.1 sequences listed in the Global Initiative (GISAID) database for sharing all influenza data have the C21772T mutation. The way 69/70 deletions cause S gene targeting damage (SGTF), and novel mutations can cause failure in PCR-based analysis.

The team performed multiple alignments and phylogenetic tree analysis to confirm that the SARS-CoV-2 infected sample was monophyletic, and the SARS-CoV-2 Wuhan-Hu-1 strain (used as a reference). When I aligned to, some alignments were misplaced. Removal of codon 69/70. Therefore, in the Nextclade and CoV Spectrum databases, the mutation was shown as A21766T (rather than C21772T).

After subjecting 17 COVID-19 samples to the Hain assay and a second vsPCR analysis for retesting, the same results were obtained with the Omicron BA.2 subvariant interpretation. After contact tracing, 10 sequences related to high school students were found, and 4 samples were epidemiologically related.

The upstream mutation A67V (C21762T) of the 69/70 deletion is normally present in the Omicron BA.1 mutant. The authors suggested that the C21772T point mutation interfered with the identification of the 69/70 codon deletion, and that the 69/70 codon deletion caused the loss of the amino acids valine (V) and histidine (H). Given that the adenine (A) -thymine (T) -cytosine (C), ATT, and ATA codons are all converted to isoleucine (I), the C21772T mutation did not cause amino acid sequence substitutions.

Conclusion

Overall, case findings indicate that the Omicron BA.1 subvariant is incorrect as the Omicron BA.2 subvariant due to a point mutation that is two nucleobases downstream of the 69/70 deletion in variant-specific PCR analysis. It was shown that it was classified. The authors believe that case reports are the first to report a C21772T mutation that causes a negative result in vsPCR analysis targeting a 69/70 deletion. The report shows that mutations in the target of the melting curve-based vs PCR assay can lead to misclassification of the SARS-CoV-2 variant. Therefore, confirming the results of the vsPCR assay with NGS may improve the accuracy of SARS-CoV-2 genome monitoring.

Several melting curve-based assays developed prior to the advent of Omicron, targeting the N501Y mutation in the SARS-CoV-2 S protein, have been applied to Omicron variant samples, probably due to the mutations surrounding amino acid 501. It gives a negative result. The recently emerging Omicron BA.4 and Omicron BA.5 subvariants show specific mutation patterns that are not expected by the assay software and require constant updates to the variant tracking software.

In addition to the SARS-CoV-2 genome surveillance challenges, A67V mutations can distinguish between Omicron BA.1 and Omicron BA.4 / 5 subvariants. However, because the Omicron BA.4 and Omicron BA.5 subvariants have similar genetic makeup at the 69 / 70del site, more targets are available to distinguish the Omicron subvariant in the vsPCR assay. Is required.

*Important Notices

Research Square publishes unpeer-reviewed preliminary scientific reports and should not be considered definitive, guide clinical / health-related behaviors, or be treated as established information. ..