Researchers report the first RT-qPCR for simultaneous detection and discrimination of Omicron sub-variants

By Bhavana KunkalikarReviewed by Danielle Ellis, B.Sc.Oct 12 2022

In a recent study published in PLOS ONE, researchers developed a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) system to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants of concern (VOCs).

Background

To stop the spread of newly discovered SARS-CoV-2 variants and develop methods for assessment of their pathogenic potential, efficient surveillance measures are required. This was crucial for the SARS-CoV-2 Omicron variant (B.1.1.529), which was predominant after the spread of the Delta variant. Particularly in nations with limited resources, RT-qPCR procedures complement whole genome sequencing. However, changes in the targeting primer and probe sequences of newly emerging variants can cause the existing RT-qPCRs to fail.

About the study

In the present study, the team developed an RT-qPCR technique that detected the SARS-CoV-2 Delta and Omicron variants simultaneously by employing a degenerate probe that specifically targeted the crucial spike protein ΔH69/V70 mutation.

TWIST Synthetic SARS-CoV-2 ribonucleic acid (RNA) control was obtained and utilized as PCR standard Alpha (B.1.17) variant in a seven-step 1:10 dilution series at a specified concentration. To test whether the RT-qPCR assays functioned with all circulating VOCs, samples were obtained from patients with whole genome sequencing (WGS)-confirmed infection with SARS-CoV-2 wild-type (WT), Alpha, Delta, or Omicron variant. In brief, community testing centers that were a part of the Danish national testing program acquired throat swabs and tested them for SARS-CoV-2 positivity.

RT-qPCR was employed to make the primary diagnosis of COVID-19. Using WGS, the identities of the lineages BA.1, BA.2, BA.4, and BA.5 were validated, and consensus genomes were deposited in Global Initiative on Sharing All Influenza Data (GISAID). Heat-inactivated Danish viral isolates corresponding to the Alpha and Delta variants cultured in Vero E6 cells were considered positive controls that took into account the main mutations (ΔH69/V70 and L452R) that were present in the Alpha, Delta, and Omicron variant. Furthermore, the cultures were isolated, cultivated, and diluted.

Two probes were created for each major mutation in the spike gene to undertake allelic discrimination analysis: one to detect the wildtype nucleotide sequence and the other to detect the mutation based on sequence alignments. The presence of SARS-CoV-2 and estimation of viral load were determined using the RT-qPCR technique that targeted the envelope (E) gene as an unspecific control.

Results

The team assessed the ΔH69/V70 RT-analytical qPCR's performance properties before including them in the Variant-PCR employed in the study for the qualitative detection of the Omicron variant in clinical samples. For the ΔH69/V70 RT-qPCR, SARS-CoV-2 Omicron, Alpha variant-infected clinical samples were used as positive controls while Wuhan and Delta-infected, as well as SARS-CoV-2 negative samples served as negative controls. Through the use of E-Sarbeco RT-qPCR multiplexed with the ΔH69/V70 RT-qPCR, the presence of SARS-CoV-2 was verified in positive samples.

Furthermore, the researchers developed a degenerate ΔH69/V70 probe to identify the ΔH69/V70 mutation present in all known SARS-CoV-2 variants. Placing the degenerate probe instead of the original one that targeted ΔH69/V70 allowed the Alpha and Omicron variants to almost fully restore their end fluorescence intensity. The absence of signal from SARS-CoV-2 variants that did not have the ΔH69/V70 deletion and the negative controls provided further evidence of the degenerate probe's specificity. A WT probe in the ΔH69/V70 RT-qPCR was employed to distinguish between the BA.1 and BA.2 sub-variants. The ΔH69/V70 WT probe successfully detected all SARS-CoV-2 variants, including WT, Delta, and Omicron BA.2, that lacked the ΔH69/V70 gene.

From each of the 745 SARS-CoV-2-positive samples, valid RT-qPCR findings and a consensus genome sequence were obtained. A total of 339 patient samples were identified as having BA.1 infections, whereas 399 samples had BA.2 infections, according to WGS analysis, indicating that the two genomes are distributed rather evenly. The remaining seven patient samples contained the Delta genome. The degenerate ΔH69/V70 probe identified the ΔH69/V70 mutation in 339 patient samples, correlating 100% with Omicron BA.1 samples.

Furthermore, a WT signal of either Delta or Omicron BA.2 origin was present in 406 clinical samples that were positive for SARS-CoV-2. Seven of these 406 samples were also found to be positive for the 452R mutation, which is typical of the Delta variation. Irrespective of the BA.1 and BA.2 sub-lineage identities, the L452-WT sequence was found in 738 of the 745 patient samples, which correlated to the Omicron variant  Altogether, the team created an RT-qPCR system for rapid Omicron BA.1 and BA.2 distinction on a wide scale, which was verified by comparing RT-qPCR and WGS data.

Conclusion

Overall, the study findings showed that by combining analysis of two distinct signature mutations in parallel, ΔH69/V70, and L452R, the team effectively detected the SARS-CoV-2 Omicron sub-lineages as well as the Delta variant.