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
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
滚环扩增(rolling circle amplification,RCA)是新近发展起来的一种恒温核酸扩增方法。这种方法不仅可以直接扩增DNA和RNA,还可以实现对靶核酸的信号放大,灵敏度达到一个拷贝的核酸分子,因此在核酸检测中具有很大的应用价值和潜力。本...滚环扩增(rolling circle amplification,RCA)是新近发展起来的一种恒温核酸扩增方法。这种方法不仅可以直接扩增DNA和RNA,还可以实现对靶核酸的信号放大,灵敏度达到一个拷贝的核酸分子,因此在核酸检测中具有很大的应用价值和潜力。本文结合了滚环扩增技术在医药领域中的最新研究进展,介绍了滚环扩增的原理及其在医药领域中的应用。展开更多
滚环扩增技术(rolling circle amplification,RCA)的建立模拟了自然界中环状病原生物DNA通过滚环模型方式自我复制的原理,经长期科学研究和实践应用,取得了诸多突破性成果。对最近几年在滚环扩增技术研究领域的最新动态进行了较全面的总...滚环扩增技术(rolling circle amplification,RCA)的建立模拟了自然界中环状病原生物DNA通过滚环模型方式自我复制的原理,经长期科学研究和实践应用,取得了诸多突破性成果。对最近几年在滚环扩增技术研究领域的最新动态进行了较全面的总结,其中包括了网状RCA、锁式探针RCA、目标成环RCA和跨越式RCA,也对滚环扩增中存在的问题进行了探讨,重点介绍了该技术在基础研究、实际检测、医疗诊断及纳米材料等方面的应用,最后对核酸等温扩增技术产业化的发展前景进行了展望。展开更多
近年来,食源性致病菌在全球范围内大面积传播,给人类的生存健康与发展带来了巨大的威胁。因此为了保证食品的安全,快速的、高特异性且高灵敏地检测食品致病菌方法已经成为科学研究的热点话题。在本研究中,我们构建了一种基于滚环扩增技...近年来,食源性致病菌在全球范围内大面积传播,给人类的生存健康与发展带来了巨大的威胁。因此为了保证食品的安全,快速的、高特异性且高灵敏地检测食品致病菌方法已经成为科学研究的热点话题。在本研究中,我们构建了一种基于滚环扩增技术的纸基显色传感器用于可视化检测致病菌的检测平台。通过滚环扩增对单增李斯特菌的hly A mRNA进行高效特异性扩增,扩增后的单链产物经过Hhal酶进行特异位点酶切后,形成与锁式探针长度相同的片段。将酶切后的单链产物不经过预变性杂交,可直接进行试纸条检测。在优化的实验条件下,可以检测到100 pg/μL总RNA。整个过程实验包括连接,扩增,酶切,检测等反应均可在恒温条件下进行,且可在几个小时内完成。这种方法适用于快速的现场检测,此外,这项研究强调了纸基显色诊断平台的潜在价值和应用前景。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。
文摘滚环扩增(rolling circle amplification,RCA)是新近发展起来的一种恒温核酸扩增方法。这种方法不仅可以直接扩增DNA和RNA,还可以实现对靶核酸的信号放大,灵敏度达到一个拷贝的核酸分子,因此在核酸检测中具有很大的应用价值和潜力。本文结合了滚环扩增技术在医药领域中的最新研究进展,介绍了滚环扩增的原理及其在医药领域中的应用。
文摘滚环扩增技术(rolling circle amplification,RCA)的建立模拟了自然界中环状病原生物DNA通过滚环模型方式自我复制的原理,经长期科学研究和实践应用,取得了诸多突破性成果。对最近几年在滚环扩增技术研究领域的最新动态进行了较全面的总结,其中包括了网状RCA、锁式探针RCA、目标成环RCA和跨越式RCA,也对滚环扩增中存在的问题进行了探讨,重点介绍了该技术在基础研究、实际检测、医疗诊断及纳米材料等方面的应用,最后对核酸等温扩增技术产业化的发展前景进行了展望。
文摘近年来,食源性致病菌在全球范围内大面积传播,给人类的生存健康与发展带来了巨大的威胁。因此为了保证食品的安全,快速的、高特异性且高灵敏地检测食品致病菌方法已经成为科学研究的热点话题。在本研究中,我们构建了一种基于滚环扩增技术的纸基显色传感器用于可视化检测致病菌的检测平台。通过滚环扩增对单增李斯特菌的hly A mRNA进行高效特异性扩增,扩增后的单链产物经过Hhal酶进行特异位点酶切后,形成与锁式探针长度相同的片段。将酶切后的单链产物不经过预变性杂交,可直接进行试纸条检测。在优化的实验条件下,可以检测到100 pg/μL总RNA。整个过程实验包括连接,扩增,酶切,检测等反应均可在恒温条件下进行,且可在几个小时内完成。这种方法适用于快速的现场检测,此外,这项研究强调了纸基显色诊断平台的潜在价值和应用前景。
