Introduction:Fluorescent probe-based recombinase aided amplification(RAA)offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design,restricting its widespre...Introduction:Fluorescent probe-based recombinase aided amplification(RAA)offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design,restricting its widespread application.Methods:A novel EvaGreen dye-based RAA(EvaGreen-RAA)assay utilizing self-avoiding molecular recognition system(SAMRS)primers was developed for the detection of Pseudomonas fluorescens(PF)and Bacillus cereus(BC)in milk.Conventional RAA was used as a reference method.Sensitivity was evaluated using nucleic acids from recombinant plasmids and simulated milk specimens.Additionally,a dual EvaGreen-RAA assay was investigated for simultaneous detection of mixed BC and PF in simulated milk specimens.Results:The EvaGreen-RAA demonstrated superior sensitivity compared to conventional RAA,with detection limits of 1 copy/μL versus 10 copies/μL for both BC and PF plasmids,respectively.In simulated milk specimens,EvaGreen-RAA detected BC and PF at concentrations of 100 CFU/mL and 200 CFU/mL,respectively,compared to 400 CFU/mL and 600 CFU/mL for conventional RAA.The dual EvaGreen-RAA assay successfully detected mixed BC and PF in simulated milk specimens at concentrations of 200 CFU/mL for each pathogen.Conclusion:The EvaGreen-RAA assay demonstrated significant advantages in terms of simplicity and enhanced sensitivity compared to fluorescent probe-based RAA,offering a novel approach for developing multiplex pathogen detection systems using melting curve analysis.展开更多
The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sort...The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sorting performance and cell viability.Here,an ultra-sensitive single-cell sorting platform has been developed by integrating the FADS technology with Tetramer-HCR-EvaGreen(THE)fluorescence signal amplification.The THE system produced much higher fluorescence signal than that of the single Tetramer or Tetramer-HCR signal amplification.Upon application to target MCF-7 cells,the platform exhibited high efficacy and selectivity while maintaining more than 95%cell viability.The THE-FADS achieved sorting efficiencies of 55.5%and 50.3%with purities of 91%and 85%for MCF-7 cells in PBS solutions and simulated serum samples,respectively.The sorted MCF-7 cells showed similar proliferation together with CK19 and EGFR mRNA expression compared with the control cells.The established THE-FADS showed the promising prospects to cellular heterogeneity understanding and personalized medicine.展开更多
基金Supported by Shandong Province Key Research and Development Plan(Major Scientific and Technological Innovation Project,2023CXGC010711)the National Key R&D Program of China(2021YFC2301102)the National Natural Science Foundation of China(82202593,U23A20106).
文摘Introduction:Fluorescent probe-based recombinase aided amplification(RAA)offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design,restricting its widespread application.Methods:A novel EvaGreen dye-based RAA(EvaGreen-RAA)assay utilizing self-avoiding molecular recognition system(SAMRS)primers was developed for the detection of Pseudomonas fluorescens(PF)and Bacillus cereus(BC)in milk.Conventional RAA was used as a reference method.Sensitivity was evaluated using nucleic acids from recombinant plasmids and simulated milk specimens.Additionally,a dual EvaGreen-RAA assay was investigated for simultaneous detection of mixed BC and PF in simulated milk specimens.Results:The EvaGreen-RAA demonstrated superior sensitivity compared to conventional RAA,with detection limits of 1 copy/μL versus 10 copies/μL for both BC and PF plasmids,respectively.In simulated milk specimens,EvaGreen-RAA detected BC and PF at concentrations of 100 CFU/mL and 200 CFU/mL,respectively,compared to 400 CFU/mL and 600 CFU/mL for conventional RAA.The dual EvaGreen-RAA assay successfully detected mixed BC and PF in simulated milk specimens at concentrations of 200 CFU/mL for each pathogen.Conclusion:The EvaGreen-RAA assay demonstrated significant advantages in terms of simplicity and enhanced sensitivity compared to fluorescent probe-based RAA,offering a novel approach for developing multiplex pathogen detection systems using melting curve analysis.
基金funded by National Key Research and Development Program of China(grant number 2022YFC3502002)CAS-NSTDA Joint Research Project(2024)(grant number 101GJHZ2024017MI).
文摘The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sorting performance and cell viability.Here,an ultra-sensitive single-cell sorting platform has been developed by integrating the FADS technology with Tetramer-HCR-EvaGreen(THE)fluorescence signal amplification.The THE system produced much higher fluorescence signal than that of the single Tetramer or Tetramer-HCR signal amplification.Upon application to target MCF-7 cells,the platform exhibited high efficacy and selectivity while maintaining more than 95%cell viability.The THE-FADS achieved sorting efficiencies of 55.5%and 50.3%with purities of 91%and 85%for MCF-7 cells in PBS solutions and simulated serum samples,respectively.The sorted MCF-7 cells showed similar proliferation together with CK19 and EGFR mRNA expression compared with the control cells.The established THE-FADS showed the promising prospects to cellular heterogeneity understanding and personalized medicine.
