Application of novel dark-quencher labeled probes in multiplex qRT-PCR assays for rapid detection of SARS-CoV-2 variants

Abstract

Background: Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the new coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because SARS-CoV-2 frequently mutates, it creates a number of variants that must be distinguished and tracked using a rapid detection technique. At present, the identification of virus variants often requires sequencing of the viral genome with sophisticated techniques which are costly and time-consuming. On the other hand, the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method used to diagnose SARS-CoV-2 infection has been widely applied worldwide amid COVID-19 pandemic. Due to the lower specificity and sensitivity in detecting different strains using multiple qRT-PCR, we aimed to develop novel dark quencher (DQ) labeled probes to improve the performance of multiple qRT-PCR. DQ probes are dihydropyrroloindole carboxylate (DPI3)-analogue.

Methods: We first tested their amplification efficiency and specificity, on detecting single nucleotide polymorphism through qRT-PCR, and the simultaneous detection efficiency of multiple SARS-CoV-2 mutation sites. The DQ labeled probes were further applied in multiplex qRT-PCR assays, and the method was validated on SARS-CoV-2 positive clinical samples for its sensitivity and specificity.

Results: DQ probes exhibited better specificity and sensitivity than the TaqMan^® Minor Groove Binder (MGB) and TaqMan probes. Great analytical sensitivity (limit of detection of 250 copies/mL), good specificity (no cross-reaction with other pathogens), and great clinical performance (99.4-100% consistency with next-generation sequencing) were demonstrated by the designed multiplex qRT-PCR tests.

Conclusions: Our novel DQ-probe/multiplex qRT-PCR assay provides a rapid and simple method to quickly distinguish SARS-CoV-2 variants, we were able to quickly identify SARS-CoV-2 variants (Delta and Omicron BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4, and BA.5) that target nine specific mutation sites in the ORF, N, NSP1, NSP3, and S genes.

Keywords: Coronavirus disease 2019 (COVID-19); dark quencher probe (DQ probe); mutation; quantitative reverse transcription-polymerase chain reaction (qRT-PCR); severe acute respiratory syndrome coronavirus 2 variants (SARS-CoV-2 variants).

Figure 1

The principle and covered viral mutations of the developed multiplex qRT-PCR assays for SARS-CoV-2 variants. (A) A novel probe named DQ; the DQ motif is DPI 3 analogue which is linked to the first base at 3' end of the target probe sequence and the structure of DPI 3 analogue. (B) Based on SARS-CoV-2 isolate Dfly-hu-1, NC_045512.2 sequences, the location of Delta and Omicron mutation sites. (C) A total of 9 mutations were enrolled in the developed assays. (D) The principle of the developed assays. The developed qRT-PCR assays consist of reactions targeting the ORF and mutation of Spike, NSP. The variation of Ct between the ORF and each mutation was employed to determine if a mutation occurred. ΔCT, CT value of each mutation site – CT value of ORF. CT, cycle threshold; DPI 3, dihydropyrroloindole carboxylate; DQ, dark quencher; N, unrecognizable; ORF, open reading frame; qRT-PCR, quantitative real-time polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; Y, identifiable.

Figure 2

Multiple mutation site detection by DQ probes and TaqMan^® MGB probes. (A) 10⁵ copies/mL of the L452R/P681H/T821I plasmid mixture and the same concentration of plasmids including the L452R/P681H/T821I single mutation were detected by DQ probes. (B) 10⁵ copies/mL of the L452R/P681H/T821I plasmid mixture and the same concentration of plasmids including the L452R/P681H/T821I single mutation were determined by TaqMan^® MGB probes. ΔRn represents the amount of probe degradation during PCR, which reflects the amount of PCR product generated. DQ, dark quencher; MGB, minor groove binder; PCR, polymerase chain reaction.

Figure 3

Amplification curve of each Omicron variant (A-Y) at 4,000, 2,000, 1,000, 500, and 250 copies/mL. Blue curve: ORF; black curve: T547K; yellow curve: P681H; light blue curve: R346K; purple curve: T842I; red curve: P681R; green curve: L452R; orange curve: 6970del; grey curve: L452Q; brown curve: KSF141-143. ΔRn represents the amount of probe degradation during PCR, which reflects the amount of PCR product generated. ORF, open reading frame; PCR, polymerase chain reaction.