Development of a panel of three multiplex allele-specific qRT-PCR assays for quick differentiation of recombinant variants and Omicron subvariants of SARS-CoV-2

Abstract

Quick differentiation of the circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmission. However, the widely used gene sequencing method is time-consuming and costly when facing the viral recombinant variants, because partial or whole genome sequencing is required. Allele-specific real time RT-PCR (qRT-PCR) represents a quick and cost-effective method in SNP genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 3 multiplex allele-specific qRT-PCR assays targeting 12 key differential mutations for quick differentiation of SARS-CoV-2 recombinant variants (XD and XE) and Omicron subvariants (BA.1 and BA.2). Two parallel multiplex qRT-PCR reactions were designed to separately target the protype allele and the mutated allele of the four mutations in each allele-specific qRT-PCR assay. The variation of Cp values (ΔCp) between the two multiplex qRT-PCR reactions was applied for mutation determination. The developed multiplex allele-specific qRT-PCR assays exhibited outstanding analytical sensitivities (with limits of detection [LoDs] of 2.97-27.43 copies per reaction), wide linear detection ranges (10⁷-10⁰ copies per reaction), good amplification efficiencies (82% to 95%), good reproducibility (Coefficient of Variations (CVs) < 5% in both intra-assay and inter-assay tests) and clinical performances (99.5%-100% consistency with Sanger sequencing). The developed multiplex allele-specific qRT-PCR assays in this study provide an alternative tool for quick differentiation of SARS-CoV-2 recombinant variants (XD and XE) and Omicron subvariants (BA.1 and BA.2).

Keywords: BA.1 subvariant; BA.2 subvariant; Omicron variant; SARS-CoV-2; allele-specific qRT-PCR; mutation; recombinant variant.

Figure 1

The principle and covered viral mutations of the developed multiplex allele-specific qRT-PCR assays. (A) The viral genomic locations of the 12 targeted mutations in the developed multiplex allele-specific qRT-PCR assays are shown, including 6 unique mutations in Omicron BA.1 subvariants (NSP3: L1266I, NSP6 I189V, Spike: S371L, Spike G446S, Spike T547K, Spike: L981F), 6 unique mutations in Omicron BA.2 subvariants (NSP1: S135R, NSP6: F108L, Spike A27S, Spike V213G, Spike R408s). The horizontal solid color lines represent the genome of the variant and subvariants of SARS-CoV-2. The vertical dashed lines indicate the boundary of viral genes. (B) The principle of the developed assays. The allele-specific qRT-PCR assays consist of two parallel reactions targeting the prototype allele and the mutated allele of each mutation, respectively. The inaccurate matched primers & probe to viral RNA generates a higher Cp value, while the accurate matched primers & probe to viral RNA generates a lower Cp value. The variation of Cp values (ΔCp) between the prototype allele targeting and the mutate allele targeting reactions was employed to determine if a mutation occurred. (C) A total of 12 mutations were enrolled in the developed assays, with 4 unique mutation patterns to identify the Omicron subvariants BA.1 and BA.2, and the recombinant variants XD and XE of SARS-CoV-2. The mutation pattern for the Omicron subvariants BA.1 and BA.2 were colored in green and red, respectively, those for the recombinant variants XD and XE were colored accordingly.

Figure 2

Determination of limits of detection for the developed multiplex allele-specific qRT-PCR assays to detect the prototype RNA of SARS-CoV-2. Limits of detection (LoDs) for the developed qRT-PCR assays to detect the prototype RNA of SARS-CoV-2 were determined by the Probit Regression (Dose-Response analysis) implemented in SPSS v22.0. qRT-PCR was performed by using the in vitro transcribed viral prototype RNA (2-fold dilution from 200 copies/reaction to 0.1 copies per reaction) as templates. The obtained Cp values and the corresponding copy number of viral RNA were applied for Probit Regression analysis. Probit curves for assay 1 [NSP1: S135R (a1), NSP6: I189V (a2), Spike: A27S (a3), and Spike: S371L (a4)], assay 2 [NSP6: F108L (b1), Spike: G446S (b2), Spike: T547K (b3) and Spike: L981F (b4)], assay 3 [NSP3: T24I (c1), NSP3: L1266I (c2), Spike: V213G (c3), and Spike: R408S (c4)] are shown. The inner solid line (in red) is a Probit curve. The outer dotted lines (in dark blue) are 95% CI.

Figure 3

Determination of limits of detection for the developed multiplex allele-specific qRT-PCR assays to detect the Mutated RNA of SARS-CoV-2. Limits of detection (LoDs) for the developed qRT-PCR assays to the mutated RNA (covering all the alleles in Table 1 ) of SARS-CoV-2 were determined by the Probit Regression (Dose-Response analysis) implemented in SPSS v22.0. qRT-PCR was performed by using the in vitro transcribed mutated RNA (2-fold dilution from 200 copies/reaction to 0.1 copies per reaction) as templates. The obtained Cp values and the corresponding copy number of viral RNA were applied for Probit Regression analysis. Probit curves for assay 1 [NSP1: S135R (a1), NSP6: I189V (a2), Spike: A27S (a3), and Spike: S371L (a4)], assay 2 [NSP6: F108L (b1), Spike: G446S (b2), Spike: T547K (b3) and Spike: L981F (b4)], assay 3 [NSP3: T24I (c1), NSP3: L1266I (c2), Spike: V213G (c3), and Spike: R408S (c4)] are shown. The inner solid line (in red) is a Probit curve. The outer dotted lines (in dark blue) are 95% CI.

Figure 4

Amplification curves for the developed multiplex allele-specific qRT-PCR assays against the prototype genes of SARS-CoV-2. The viral prototype genes (NSP1, NSP3, NSP6 and Spike) were in vitro transcribed and 10-fold diluted from 10⁷ copies/reaction (2.33×10⁷, 4.45×10⁷, 3.10×10⁷, 6.82×10⁷ for NSP1, NSP3, NSP6 and Spike, respectively) to 10^-1 copies/reaction. Two parallel reactions targeting the prototype allele and the mutated allele of the developed assays were performed by using the in vitro transcribed and serially diluted prototype RNA as template. The amplification curves for the prototype allele targeting reactions (in dark blue) were then merged with those of the corresponding mutated allele targeting reactions (in red). The amplification curves for assay 1 [NSP1: S135R (a1), NSP6: I189V (a2), Spike: A27S (a3), and Spike: S371L (a4)], assay 2 [NSP6: F108L (b1), Spike: G446S (b2), Spike: T547K (b3) and Spike: L981F (b4)], assay 3 [NSP3: T24I (c1), NSP3: L1266I (c2), Spike: V213G (c3), and Spike: R408S (c4)] are shown.

Figure 5

Amplification curves for the developed multiplex allele-specific qRT-PCR assays against the mutated genes of SARS-CoV-2. The viral mutated genes (NSP1, NSP3, NSP6 and Spike) covering all the mutations listed in Table 1 were in vitro transcribed and 10-fold diluted from 10⁷ copies/reaction (8.59×10⁷, 8.83×10⁷, 6.92×10⁷, 2.45×10⁷ for NSP1, NSP3, NSP6 and Spike, respectively) to 10^-1 copies/reaction. Two parallel reactions targeting the prototype allele and the mutated allele of the developed assays were performed by using the in vitro transcribed and serially diluted mutated RNA as template. The amplification curves for the mutated allele targeting reactions (in red) were then merged with those of the corresponding prototype allele targeting reactions (in dark blue). The amplification curves for assay 1 [NSP1: S135R (a1), NSP6: I189V (a2), Spike: A27S (a3), and Spike: S371L (a4)], assay 2 [NSP6: F108L (b1), Spike: G446S (b2), Spike: T547K (b3) and Spike: L981F (b4)], assay 3 [NSP3: T24I (c1), NSP3: L1266I (c2), Spike: V213G (c3), and Spike: R408S (c4)] are shown.

Figure 6

Evaluation of cross-reactivity between the developed assays and common respiratory viruses and pathogenic bacteria. The nucleic acids from the other coronaviruses (including NL63, OC43 and 229E), other respiratory viruses (including influenza viruses, parainfluenza viruses, and respiratory syncytial virus) and pathogenic bacteria (including Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus and Pseudomonas aeruginosa) were used as templates (n=16) to check the cross-reactivity between the developed assays and common respiratory pathogens. The amplification curves for assay 1 [NSP1: S135R (a1), NSP6: I189V (a2), Spike: A27S (a3), and Spike: S371L (a4)], assay 2 [NSP6: F108L (b1), Spike: G446S (b2), Spike: T547K (b3) and Spike: L981F (b4)], assay 3 [NSP3: T24I (c1), NSP3: L1266I (c2), Spike: V213G (c3), and Spike: R408S (c4)] are shown.