The recent pneumonia outbreak caused by a novel coronavirus(SARS-CoV-2)is posing a great threat to global public health.Therefore,rapid and accurate identification of pathogenic viruses plays a vital role in selecting...The recent pneumonia outbreak caused by a novel coronavirus(SARS-CoV-2)is posing a great threat to global public health.Therefore,rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments,saving people's lives and preventing epidemics.It is important to establish a quick standard diagnostic test for the detection of the infectious disease(COVID-19)to prevent subsequent secondary spread.Polymerase chain reaction(PCR)is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity.Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler opera-tion.A variety of improved or new approaches also have been developed.This review summarizes the currently available detection methods for coronavirus nucleic acid.It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coro-navirus infection.展开更多
Comprehensive Summary,Most conventional digital bioassays rely on the use of fully-sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction,which require sp...Comprehensive Summary,Most conventional digital bioassays rely on the use of fully-sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction,which require specialized equipment or proprietary reagents/consumables.Herein,we report a microchamber-free and spherical nucleic acid(SNA)-amplified digital flow cytometric bead assay(dFBA)for ultrasensitive protein and exosome analysis with simple workflows,easily accessible instruments/reagents,and high discriminating ability towards the fluorescence-positive and fluorescence-negative beads.In this dFBA,microbeads are employed as independent carriers to anchor the single target molecule-initiated signal amplification reaction,avoiding the use of sealed droplets or microwell microchambers.Meanwhile,antibody-functionalized SNAs(FSNAs)with a high density of DNA probes act as a bridge for efficiently amplified target-to-DNA signal conversion,which allows the use of DNA-based rolling circle amplification(RCA)as the fluorescence signal amplification technique to quantify non-nucleic acid targets.Even a single target-induced on-bead RCA and fluorescence enriching are sufficient to make the target-loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer(FCM).This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers,enlarging the toolbox of digital bioassays for clinical applications.展开更多
基金financial support from the National Natural Science Foundation of China(Grant 81973281)the Fundamental Research Funds for the Central Universities(2019FZA7017)Leading Talent of“Ten Thousand Plan”-National High-Level Talents SpecialSupport Plan。
文摘The recent pneumonia outbreak caused by a novel coronavirus(SARS-CoV-2)is posing a great threat to global public health.Therefore,rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments,saving people's lives and preventing epidemics.It is important to establish a quick standard diagnostic test for the detection of the infectious disease(COVID-19)to prevent subsequent secondary spread.Polymerase chain reaction(PCR)is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity.Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler opera-tion.A variety of improved or new approaches also have been developed.This review summarizes the currently available detection methods for coronavirus nucleic acid.It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coro-navirus infection.
基金supported by the National Natural Science Foundation of China(22074088,21622507)the Program for Changjiang Scholars and Innovative Research Team in University(IRT_15R43)+1 种基金the Fundamental Research Funds for the Central Universities(GK202101001,GK202206040)Innovation Capability Support Program of Shaanxi(2021TD-42).
文摘Comprehensive Summary,Most conventional digital bioassays rely on the use of fully-sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction,which require specialized equipment or proprietary reagents/consumables.Herein,we report a microchamber-free and spherical nucleic acid(SNA)-amplified digital flow cytometric bead assay(dFBA)for ultrasensitive protein and exosome analysis with simple workflows,easily accessible instruments/reagents,and high discriminating ability towards the fluorescence-positive and fluorescence-negative beads.In this dFBA,microbeads are employed as independent carriers to anchor the single target molecule-initiated signal amplification reaction,avoiding the use of sealed droplets or microwell microchambers.Meanwhile,antibody-functionalized SNAs(FSNAs)with a high density of DNA probes act as a bridge for efficiently amplified target-to-DNA signal conversion,which allows the use of DNA-based rolling circle amplification(RCA)as the fluorescence signal amplification technique to quantify non-nucleic acid targets.Even a single target-induced on-bead RCA and fluorescence enriching are sufficient to make the target-loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer(FCM).This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers,enlarging the toolbox of digital bioassays for clinical applications.
