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
The Anxin brand burglar-proof alarm padlock is manufactured by the Shanghai New Star Special Equipment Company. It has the combined effect of locking, alarm and guarding against theft.
依据对虾黄头病毒（Yellow head virus,YHV）的非结构蛋白N基因序列,设计特异的锁式探针（Padlock probe,PLP）、检测探针及引物,建立YHV超分支滚环扩增（Hyper-branched rolling circle amplification,HRCA）检测试纸。灵敏度实验显示,...依据对虾黄头病毒（Yellow head virus,YHV）的非结构蛋白N基因序列,设计特异的锁式探针（Padlock probe,PLP）、检测探针及引物,建立YHV超分支滚环扩增（Hyper-branched rolling circle amplification,HRCA）检测试纸。灵敏度实验显示,YHV HRCA检测试纸能检测出的最低模板量为101拷贝,是RT-PCR灵敏度的100倍。特异性实验结果表明,该试纸能够特异性地对YHV进行检测。利用该检测试纸对进出口80批次虾样本进行检测,并将检测结果与常规RT-PCR相比较,结果显示,YHV HRCA检测试纸灵敏度方面优于常规RT-PCR方法,且操作简便、结果直观易读。展开更多
依据传染性皮下及造血组织坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)的非结构蛋白NS1基因序列,设计特异的锁式探针,建立IHHNV病毒超分支滚环扩增(hyper-branched rolling circle amplification,HRCA)检...依据传染性皮下及造血组织坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)的非结构蛋白NS1基因序列,设计特异的锁式探针,建立IHHNV病毒超分支滚环扩增(hyper-branched rolling circle amplification,HRCA)检测方法并构建试纸条.结果表明,IHHNV HRCA试纸的检测限可以达到10拷贝/μL,较常规PCR法高约2个数量级,且能够保证对IHHNV的特异性检测.利用该试纸条对国产与进口的42份虾样本进行IHHNV检测,结果显示,HRCA试纸条在灵敏度上优于常规PCR,且方法直观、更易于结果判定.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The Anxin brand burglar-proof alarm padlock is manufactured by the Shanghai New Star Special Equipment Company. It has the combined effect of locking, alarm and guarding against theft.
文摘依据对虾黄头病毒（Yellow head virus,YHV）的非结构蛋白N基因序列,设计特异的锁式探针（Padlock probe,PLP）、检测探针及引物,建立YHV超分支滚环扩增（Hyper-branched rolling circle amplification,HRCA）检测试纸。灵敏度实验显示,YHV HRCA检测试纸能检测出的最低模板量为101拷贝,是RT-PCR灵敏度的100倍。特异性实验结果表明,该试纸能够特异性地对YHV进行检测。利用该检测试纸对进出口80批次虾样本进行检测,并将检测结果与常规RT-PCR相比较,结果显示,YHV HRCA检测试纸灵敏度方面优于常规RT-PCR方法,且操作简便、结果直观易读。
文摘依据传染性皮下及造血组织坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)的非结构蛋白NS1基因序列,设计特异的锁式探针,建立IHHNV病毒超分支滚环扩增(hyper-branched rolling circle amplification,HRCA)检测方法并构建试纸条.结果表明,IHHNV HRCA试纸的检测限可以达到10拷贝/μL,较常规PCR法高约2个数量级,且能够保证对IHHNV的特异性检测.利用该试纸条对国产与进口的42份虾样本进行IHHNV检测,结果显示,HRCA试纸条在灵敏度上优于常规PCR,且方法直观、更易于结果判定.
