tetano
Editor, Senior Moderator
J Thorac Dis
. 2025 Apr 30;17(4):2159-2173.
doi: 10.21037/jtd-24-853. Epub 2025 Apr 28. Application of novel dark-quencher labeled probes in multiplex qRT-PCR assays for rapid detection of SARS-CoV-2 variants
Zhiqi Zeng[SUP] #[/SUP][SUP] 1 2 3 4 [/SUP], Jie Yang[SUP] #[/SUP][SUP] 5 [/SUP], Jun Dai[SUP] #[/SUP][SUP] 6 [/SUP], Lirong Zou[SUP] #[/SUP][SUP] 7 [/SUP], Zhengshi Lin[SUP] #[/SUP][SUP] 1 [/SUP], Yong Liu[SUP] #[/SUP][SUP] 2 5 [/SUP], Wenda Guan[SUP] #[/SUP][SUP] 1 [/SUP], Feng Li[SUP] #[/SUP][SUP] 8 [/SUP], Kui Zheng[SUP] 6 [/SUP], Shuai Yuan[SUP] 6 [/SUP], Fangfang Sun[SUP] 6 [/SUP], Fengxia He[SUP] 6 [/SUP], Ye Hong[SUP] 6 [/SUP], Hui Li[SUP] 9 [/SUP], Wei Liu[SUP] 9 [/SUP], Guangqi Men[SUP] 9 [/SUP], Xinyue Zhang[SUP] 9 [/SUP], Yun Lan[SUP] 8 [/SUP], Xizi Deng[SUP] 8 [/SUP], Liya Li[SUP] 8 [/SUP], Yaqing Lin[SUP] 8 [/SUP], Honghao Lai[SUP] 8 [/SUP], Peng Qian[SUP] 8 [/SUP], Qinghong Fan[SUP] 8 [/SUP], Mengling Jiang[SUP] 8 [/SUP], Jiaojiao Li[SUP] 8 [/SUP], Guofang Tang[SUP] 8 [/SUP], Qiaohui Mo[SUP] 2 [/SUP], Xiaoyan Deng[SUP] #[/SUP][SUP] 2 3 4 [/SUP], Jicheng Huang[SUP] #[/SUP][SUP] 6 [/SUP], Xiaoling Deng[SUP] #[/SUP][SUP] 7 [/SUP], Zifeng Yang[SUP] #[/SUP][SUP] 1 2 3 4 10 [/SUP]
Affiliations
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[SUP]®[/SUP] 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).
. 2025 Apr 30;17(4):2159-2173.
doi: 10.21037/jtd-24-853. Epub 2025 Apr 28. Application of novel dark-quencher labeled probes in multiplex qRT-PCR assays for rapid detection of SARS-CoV-2 variants
Zhiqi Zeng[SUP] #[/SUP][SUP] 1 2 3 4 [/SUP], Jie Yang[SUP] #[/SUP][SUP] 5 [/SUP], Jun Dai[SUP] #[/SUP][SUP] 6 [/SUP], Lirong Zou[SUP] #[/SUP][SUP] 7 [/SUP], Zhengshi Lin[SUP] #[/SUP][SUP] 1 [/SUP], Yong Liu[SUP] #[/SUP][SUP] 2 5 [/SUP], Wenda Guan[SUP] #[/SUP][SUP] 1 [/SUP], Feng Li[SUP] #[/SUP][SUP] 8 [/SUP], Kui Zheng[SUP] 6 [/SUP], Shuai Yuan[SUP] 6 [/SUP], Fangfang Sun[SUP] 6 [/SUP], Fengxia He[SUP] 6 [/SUP], Ye Hong[SUP] 6 [/SUP], Hui Li[SUP] 9 [/SUP], Wei Liu[SUP] 9 [/SUP], Guangqi Men[SUP] 9 [/SUP], Xinyue Zhang[SUP] 9 [/SUP], Yun Lan[SUP] 8 [/SUP], Xizi Deng[SUP] 8 [/SUP], Liya Li[SUP] 8 [/SUP], Yaqing Lin[SUP] 8 [/SUP], Honghao Lai[SUP] 8 [/SUP], Peng Qian[SUP] 8 [/SUP], Qinghong Fan[SUP] 8 [/SUP], Mengling Jiang[SUP] 8 [/SUP], Jiaojiao Li[SUP] 8 [/SUP], Guofang Tang[SUP] 8 [/SUP], Qiaohui Mo[SUP] 2 [/SUP], Xiaoyan Deng[SUP] #[/SUP][SUP] 2 3 4 [/SUP], Jicheng Huang[SUP] #[/SUP][SUP] 6 [/SUP], Xiaoling Deng[SUP] #[/SUP][SUP] 7 [/SUP], Zifeng Yang[SUP] #[/SUP][SUP] 1 2 3 4 10 [/SUP]
Affiliations
- PMID: 40400917
- PMCID: PMC12090148
- DOI: 10.21037/jtd-24-853
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[SUP]®[/SUP] 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).