Molecular detection of SARS-CoV-2 strains and differentiation of Delta variant strains

Abstract

The Delta variant of SARS-CoV-2 has now become the predominant strain in the global COVID-19 pandemic. Strain coverage of some detection assays developed during the early pandemic stages has declined due to periodic mutations in the viral genome. We have developed a real-time RT-PCR (RT-qPCR) for SARS-CoV-2 detection that provides nearly 100% strain coverage, and differentiation of highly transmissible Delta variant strains. All full or nearly full (≥28 kb) SARS-CoV-2 genomes (n = 403,812), including 6422 Delta and 280 Omicron variant strains, were collected from public databases at the time of analysis and used for assay design. The two amino acid deletions in the spike gene (S-gene, Δ156-157) that is characteristic of the Delta variant were targeted during the assay design. Although strain coverage for the Delta variant was very high (99.7%), detection coverage for non-Delta wild-type strains was 93.9%, mainly due to the confined region of design. To increase strain coverage of the assay, the design for CDC N1 target was added to the assay. In silico analysis of 403,812 genomes indicated a 95.4% strain coverage for the CDC N1 target, however, in combination with our new non-Delta S-gene target, total coverage for non-Delta wild-type strains increased to 99.8%. A human 18S rRNA gene was also analyzed and used as an internal control. The final four-plex RT-qPCR assay generated PCR amplification efficiencies between 95.4% and 102.0% with correlation coefficients (R² ) of >0.99 for cloned positive controls; Delta and non-Delta human clinical samples generated PCR efficiencies of 93.4%-97.0% and R² > 0.99. The assay also detects 98.6% of 280 Omicron sequences. Assay primers and probes have no match to other closely related human coronaviruses, and did not produce a signal from samples positive to selected animal coronaviruses. Genotypes of selected clinical samples identified by the RT-qPCR were confirmed by Sanger sequencing.

Keywords: COVID-19; Delta variant; Omicron variant; PCR; SARS-CoV-2; assay; diagnosis.

Figure 1.

Alignment of primers and probes of this assay with 5 strains each from the Delta and non-Delta strains of SARS-CoV-2, SARS-CoV-1, and MERS (Middle East Respiratory Syndrome coronavirus), and other human coronavirus HKU1, OC43, NL63 and 229E strains. SARS2-dF: forward primer for the RT-qPCR; Reverse primer SARS2-dR, and probes SARS2-dPr and SARS2-wPr are shown as reverse complimentary sequences; “.” indicates same nucleotide to the reference sequence, SARS2-W|OK426939.1; “-” indicates missing nucleotide.

Figure 2:

Multiplex RT-qPCR standard curve analysis of cloned plasmids. SARS2-d: S-gene assay for Delta variant strains; SARS2-w: S-gene assay for non-Delta wildtype strains; nCoV-N1: CDC N-gene assay; 18S-3: human 18S ribosomal RNA (18S rRNA) gene as internal control.

Figure 3:

Standard curve analysis of Delta variant clinical sample. SARS2-d: S-gene assay for Delta variant strains; nCoV-N1: CDC N-gene assay. There was no data generated by the non-Delta variant probe, SARS2-w.

Figure 4:

Standard curve analysis of wildtype strain clinical sample. SARS2-w: S-gene assay for non-Delta wildtype strains; nCoV-N1: CDC N-gene assay. There is no data generated on Delta variant probe, SARS2-d.

Figure 5:

Visual representation of the S gene region encompassing the 468–473 nt deleted region (corresponding to Δ156–157 aa deletion) in 10 selected Delta variant strains, and compared to 10 selected non-Delta wildtype strains. An annotated Delta variant and a non-Delta wildtype sequences collected from NCBI were also used in the sequence comparison. “.” Indicates same nucleotide to the reference sequence, MZ636302.1, listed on top of the chart; “-”. Indicates missing nucleotide.