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

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.