Selection of variant specific gene mutations
Apart from the universal SARS-CoV-2 target RdRP, various reports regarding variant specific regions have been identified. Regions distinctly mutated in the Delta variant are the T19R, L452R and P681R region 16. The mutation percentage of each gene specific to the variant were estimated around 98.3, 97.1, and 99.2 % (Table 1). One of the Omicron specifically mutated genes are known to be T547K, D796Y and Q954H 19. The percentage was also very high ranging from 94 to 98 %. These distinct features indicate if primers can pinpoint at least two mutations of any variant, it would designate the patient to be diagnosed as Gamma or Omicron variant SARS-CoV-2. Alongside selecting the genes, SNP regions of each target were selected for optimal primer assembling (Sup Fig 1). To enhance the efficiency of the overall SARS-CoV-2 detection process, four distinct colored fluorophores were used to effectively distinguish PCR amplification between SARS-CoV-2, Delta and Omicron variant, and negative control. To decrease false negative results, each fluorophores consisted of probes matching to three point mutations of each variant. To confirm whether two specific regions of each variant could represent a positive match, correlation analysis was done for mutation of three genes T19R, L452R and P681R in Delta variants, T547K, D796Y and Q954H in Omicron variants. Results showed a 99.58%
correlation of three variants co-existing in Delta variants, and 99.48% in omicron variants (Table 2). This validates when the two of three mutated regions of each variant were matched, it would indicate a positive diagnose of the patient as Delta or Omicron SARS-CoV-2.
Optimization of the PCR procedure
Once the primer targets were set, multiple PCR tests were performed for optimal amplification. All PCR tests were evaluated using standard plasmid DNAs containing inserts corresponding target regions. Amplification of RdRp and Negative control was initially confirmed to rule out possible interference. As suspected, all SARS-CoV-2 variants tested showed high amplified signals of RdRp and no signals of negative controls (Fig 1). PCR trials of Delta and Omicron variants were also performed. For each variant, every primer/probe set was tested with the SARS-CoV-2 positive and negative controls to ensure every amplified signal was detected over the PCR threshold. As a result, amplification signals of T19R, L452R, P681R within the Delta variant and T547K, D796Y, Q954H within the Omicron variant was detected above the PCR threshold (Fig 1). After the validation of each target, three targets altogether of each variant were tested. The detection signal was as strong as the positive control RdRp and was easily distinguishable to be selected as a match. Finally, to confirm the variants were only amplified in a SNP mutated primer, wildtype standard plasmid of each target was tested. As a result, each primer sets for the Delta and Omicron variants did not amplify using the wildtype plasmid DNA as a template, proving no interference of non-specific template annealing.
Validation using RNA samples of SARS-CoV-2 patients
Once the optimization was done, further validation was done using donated RNA extraction of SARS-CoV-2 patients. Each RNA samples were tested with primer/probe targets of T19R, L452R, P681R within the Delta variant and T547K, D796Y, Q954H within the Omicron variant. When comparing the amplified data with the plasmid DNA, individual target signals were in par with the RdRp positive control (Fig 2). When all three primer sets were tested in a single PCR, amplified signals of Delta and Omicron variants were far above the positive control. To further test the amplification specificity, sufficient amount of Covid RNA was included in each PCR validation. When the input was increased to 5 x 107 RNA of both variants, amplified signals surpassed RdRp positive control (Fig 2C). The results would mean actual patient PCR trials will be more easily diagnosed.