Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assist...Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assisted methods are important approaches for RNA direct detection,but its specificity will be limited when the fidelity of ligases is not ideal.The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection.Methods In this study,a dualcompetitive-padlock-probe(DCPLP)assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation.To verify the method,we employed dual competitive padlock probe-mediated rolling circle amplification(DCPLP-RCA)to genotype the CYP2C9 gene.Results The specificity was well improved through the competition and strand displacement of dual padlock probe,with an 83.26%reduction in nonspecific signal.By detecting synthetic RNA samples,the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L.Furthermore,clinical samples were applied to the method to evaluate its performance,and the genotyping results were consistent with those obtained using the qPCR method.Conclusion This study has successfully established a highly specific direct RNA SNP detection method,and provided a novel avenue for accurate identification of various types of RNAs.展开更多
In this study,a self-calibrating near-infrared fluorescence probe was designed and synthesized based on the dual-fluorophore strategy utilizing methylene blue and coumarin.The probe utilized methylene blue(emission sp...In this study,a self-calibrating near-infrared fluorescence probe was designed and synthesized based on the dual-fluorophore strategy utilizing methylene blue and coumarin.The probe utilized methylene blue(emission spectrum range:640-740 nm)and coumarin fluorophore(emission spectrum range:440-600 nm)as signal output units,thereby achieving effective spectral separation and highly selective detection of HClO.Under physiological pH conditions,HClO triggers an oxidation-cleavage reaction,releasing methylene blue and coumarin,which emit distinct red and green fluorescence,respectively.This dual-emission feature enabled rapid HClO detection with two-channel detection limits of 25.13 nmol·L^(-1)(green channel)and 31.55 nmol·L^(-1)(red channel).Furthermore,in cell imaging experiments,this probe demonstrated excellent cell membrane permeability and low cytotoxicity,successfully enabling the monitoring of both endogenous and exogenous HClO in living cells.By incorporating a twochannel self-calibration system,the probe effectively mitigated signal variations caused by instrumental or environmental interference,substantially improving detection sensitivity and reliability.展开更多
文摘Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assisted methods are important approaches for RNA direct detection,but its specificity will be limited when the fidelity of ligases is not ideal.The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection.Methods In this study,a dualcompetitive-padlock-probe(DCPLP)assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation.To verify the method,we employed dual competitive padlock probe-mediated rolling circle amplification(DCPLP-RCA)to genotype the CYP2C9 gene.Results The specificity was well improved through the competition and strand displacement of dual padlock probe,with an 83.26%reduction in nonspecific signal.By detecting synthetic RNA samples,the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L.Furthermore,clinical samples were applied to the method to evaluate its performance,and the genotyping results were consistent with those obtained using the qPCR method.Conclusion This study has successfully established a highly specific direct RNA SNP detection method,and provided a novel avenue for accurate identification of various types of RNAs.
文摘In this study,a self-calibrating near-infrared fluorescence probe was designed and synthesized based on the dual-fluorophore strategy utilizing methylene blue and coumarin.The probe utilized methylene blue(emission spectrum range:640-740 nm)and coumarin fluorophore(emission spectrum range:440-600 nm)as signal output units,thereby achieving effective spectral separation and highly selective detection of HClO.Under physiological pH conditions,HClO triggers an oxidation-cleavage reaction,releasing methylene blue and coumarin,which emit distinct red and green fluorescence,respectively.This dual-emission feature enabled rapid HClO detection with two-channel detection limits of 25.13 nmol·L^(-1)(green channel)and 31.55 nmol·L^(-1)(red channel).Furthermore,in cell imaging experiments,this probe demonstrated excellent cell membrane permeability and low cytotoxicity,successfully enabling the monitoring of both endogenous and exogenous HClO in living cells.By incorporating a twochannel self-calibration system,the probe effectively mitigated signal variations caused by instrumental or environmental interference,substantially improving detection sensitivity and reliability.
