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Application of novel dark-quencher labeled probes in multiplex qRT-PCR assays for rapid detection of SARS-CoV-2
Zhiqi Zeng1,2,3,4, Jie Yang5, Jun Dai6
1State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
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.
Insights
Novel dark quencher (DQ) probes enhance multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays for rapid SARS-CoV-2 variant detection. This method offers improved specificity and sensitivity for identifying COVID-19 strains.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frequently mutates, necessitating rapid detection techniques for distinguishing variants.
- Current genomic sequencing methods for variant identification are costly and time-consuming.
- Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is widely used for SARS-CoV-2 detection but faces limitations in specificity and sensitivity for different strains.
Purpose of the Study:
- To develop novel dark quencher (DQ) labeled probes to enhance the performance of multiplex qRT-PCR.
- To improve the specificity and sensitivity of qRT-PCR for detecting and distinguishing SARS-CoV-2 variants.
Main Methods:
- Novel DQ probes, analogues of dihydropyrroloindole carboxylate (DPI3), were designed and synthesized.
- The amplification efficiency and specificity of DQ probes were tested using qRT-PCR for single nucleotide polymorphism detection and simultaneous detection of multiple SARS-CoV-2 mutation sites.
- The DQ-probe-based multiplex qRT-PCR assay was validated using SARS-CoV-2 positive clinical samples.
Main Results:
- DQ probes demonstrated superior specificity and sensitivity compared to TaqMan® Minor Groove Binder (MGB) and TaqMan probes.
- The multiplex qRT-PCR assay achieved high analytical sensitivity (limit of detection of 250 copies/mL) and specificity (no cross-reaction with other pathogens).
- Clinical validation showed excellent performance, with 99.4-100% consistency compared to next-generation sequencing.
Conclusions:
- The developed DQ-probe/multiplex qRT-PCR assay offers a rapid, simple, and effective method for distinguishing SARS-CoV-2 variants.
- The assay successfully identified multiple SARS-CoV-2 variants, including Delta and various Omicron sublineages, by targeting specific mutation sites in key genes (ORF, N, NSP1, NSP3, S).
