A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA ...A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA （ssDNA） were spotted on a microarray. The endonuclease recognition site （ERS） and the DNA-binding sites （DBS） were arranged side by side within the duplex region. The working principle of the detection system is described as follows： when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification （RCA）. When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.展开更多
Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The...Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The success of BSV management is inherently coupled to the availability of a sensitive indexing method. In this study, the sensitivity of three BSV detection techniques: rolling circle amplification (RCA), immunocapture PCR (with degenerate and Gold finger primers) and standard PCR was compared. A set of 32 BSD-asymptomatic samples were used to compare the techniques. Analysis of variance (ANOVA) for comparison of the four techniques showed that there were significant differences (P Musa tissues for BSV. This study unveils a more reliable BSV detection method, a need that has remained unaddressed for a long while.展开更多
Cyclic polyolefin (COP) is an inexpensive hydrophobic material with low auto-fluorescence, high light transmittance and thermal stability, broad chemical resistance and no non-specific protein binding. Here, the hydro...Cyclic polyolefin (COP) is an inexpensive hydrophobic material with low auto-fluorescence, high light transmittance and thermal stability, broad chemical resistance and no non-specific protein binding. Here, the hydrophobic alkane COP was modified to have carbonyl functionalities through oxygen plasma and chemical etching treatments to increase usefulness for chemical and biochemical applications. Then, biotin-hydrazide was used to create biotinylated surfaces that bound streptavidin. A biotinylated target oligonucleotide was subsequently bound to the immobilized biotin-streptavidin and ligation mediated rolling circle amplification-based (L-RCA) SNP detection was demonstrated.展开更多
为可视化检测海产品中的活副溶血性弧菌,该研究将叠氮溴化丙锭(propidium monoazide,PMA)染料与跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)技术相结合,以toxR为靶基因,设计并筛选引物,成功建立可视化PMA⁃SRCA检...为可视化检测海产品中的活副溶血性弧菌,该研究将叠氮溴化丙锭(propidium monoazide,PMA)染料与跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)技术相结合,以toxR为靶基因,设计并筛选引物,成功建立可视化PMA⁃SRCA检测方法,并评估所建立的方法。研究表明,12株副溶血性弧菌样品呈现阳性,29株非副溶血性弧菌样品呈现阴性,表明该方法特异性良好。该方法灵敏度为3.2×100 CFU/mL,高于可视化PMA⁃环介导等温扩增(3.2×101 CFU/mL)和可视化PMA⁃聚合酶链式反应(3.2×102 CFU/mL)的灵敏度。对76份市售海鲜样品进行检测,该方法的敏感性为100.00%,特异性为92.00%,符合率为97.37%。综上所述,可视化PMA⁃SRCA检测方法可检测活的副溶血性弧菌,具有良好的特异性、较优异的灵敏度。展开更多
将生物芯片技术和滚环扩增(rolling circle amplification,RCA)技术结合,建立一种在片RCA检测食源致病性菌新方法.依据待检测致病菌的基因组序列设计捕获探针(capture probe,CP)、检测探针(detect probe,DP)和滚环探针(rolling circle p...将生物芯片技术和滚环扩增(rolling circle amplification,RCA)技术结合,建立一种在片RCA检测食源致病性菌新方法.依据待检测致病菌的基因组序列设计捕获探针(capture probe,CP)、检测探针(detect probe,DP)和滚环探针(rolling circle probe,RCP),其中捕获探针、检测探针与待检测致病菌基因组序互补,RCP不含任何待检菌基因组DNA序列,CP的5′末端修饰氨基,将其点样制备成CP微阵列,DP、RCP与待检菌gDNA混合后变性,将其与CP微阵列杂交并连接, RCA扩增反应体系中加入链亲和素进行在片扩增反应,分析检测结果.研究表明,该方法能灵敏、特异性地检测单一和混合靶标分子,以金黄色葡萄球菌为检测对象,其最低检测限可到达50 fg/μL,其线性范围是400 ~50 fg/μL, R 2 达到0.97,证实了该方法的可行性和可靠性,为食源性致病菌检测提供了新的技术方法.展开更多
目的:应用滚环扩增(rolling circle amplification,RCA)技术以DNA芯片为载体建立一种对结核分枝杆菌耐药基因单碱基突变的快速检测方法。方法:根据结核分枝杆菌利福平耐药rpoB基因序列,设计针对该基因常见突变位点的锁式探针,及固定于...目的:应用滚环扩增(rolling circle amplification,RCA)技术以DNA芯片为载体建立一种对结核分枝杆菌耐药基因单碱基突变的快速检测方法。方法:根据结核分枝杆菌利福平耐药rpoB基因序列,设计针对该基因常见突变位点的锁式探针,及固定于基因芯片上的捕获探针。针对临床结核分枝杆菌样本的基因组DNA,PCR扩增含有rpoB基因常见突变位点的特异性DNA片段。将与突变型特异性互补结合并环化的锁式探针与芯片上固定的捕获探针进行杂交,并运用滚环扩增技术,将含有生物素标记的dUTP掺入扩增产物,最后通过与亲和素标记的纳米金反应,并银染增强显色。同时与临床样本的测序结果比较。结果:通过优化反应条件,能特异性的检测出结核分枝杆菌耐利福平rpoB基因的单碱基突变,通过对临床样本的检测,结果与测序结果一致。结论:该方法结合了DNA芯片和滚环扩增技术,能够快速有效的检测出耐药结核的单碱基突变,具有高特异性、高灵敏度。展开更多
Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,c...Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.展开更多
该文构建一种基于靶标诱导滚环扩增(rolling circle amplification,RCA)的无标记适配体快速检测赭曲霉毒素A(ochratoxin A,OTA)生物传感器。该生物传感器探针由RCA引物与OTA适配体两部分组成,在OTA存在的环境中,OTA适配体特异性识别靶标...该文构建一种基于靶标诱导滚环扩增(rolling circle amplification,RCA)的无标记适配体快速检测赭曲霉毒素A(ochratoxin A,OTA)生物传感器。该生物传感器探针由RCA引物与OTA适配体两部分组成,在OTA存在的环境中,OTA适配体特异性识别靶标,探针结构被打开,RCA引物与环状DNA模板(circular DNA template,CT)结合开启RCA反应,加入核酸染料SYBR Gold产生荧光信号。此生物传感器具有较高的特异性,检测限为6.6×10^(-2)nmol/L,线性检测范围为6.6×10^(-2)~660 nmol/L,可用于具体的分析检测。此生物传感器无需复杂化学修饰且操作简单,在食品安全检测中具有良好的应用前景。展开更多
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
Compared with other types of breast cancer,triple-negative breast cancer(TNBC)has the characteristics of a high degree of malignancy and poor prognosis.Early diagnosis of TNBC through biological markers and timely dev...Compared with other types of breast cancer,triple-negative breast cancer(TNBC)has the characteristics of a high degree of malignancy and poor prognosis.Early diagnosis of TNBC through biological markers and timely development of effective treatment methods can reduce its mortality.Many Research experiments have confirmed that some specific mi RNA expression profiles in TNBC can used as markers for early diagnosis.However,detecting the expression profiles of multiple groups of miRNAs according to traditional detection methods is complicated and consumes many samples.To address this issue,we developed a method for high-throughput,high-sensitivity quantitative detection of multiple sets of miRNAs(including mi R-16,mi R-21,mi R-92,mi R-199,and mi R-342)specifically expressed in TNBC by rolling circle amplification(RCA)on fluorescence-encoded microspheres.Through the optimization of reaction system conditions,the developed method showed an extensive linear dynamic range and high sensitivity for all five miRNAs with the lowest limit of detection of 2 fmol/L.Meanwhile,this high-throughput detection method also appeared reasonable specificity.Only in the presence of a specific target miRNA,the fluorescence signal on the correspondingly encoded microspheres is significantly increased,while the fluorescence signal on other non-correspondingly encoded microspheres is almost negligible.Furthermore,this process exhibited good recovery and reproducibility in serum.The advantages of this method allow us to more conveniently obtain the expression profiles of multiple groups of TNBC-associated mi RNAs,which is beneficial for the early detection of TNBC.展开更多
A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and te...A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.展开更多
基金supported by the National Natural Science Foundation of China(Nos.60501010,60701008 and 60771024)
文摘A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA （ssDNA） were spotted on a microarray. The endonuclease recognition site （ERS） and the DNA-binding sites （DBS） were arranged side by side within the duplex region. The working principle of the detection system is described as follows： when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification （RCA）. When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.
文摘Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The success of BSV management is inherently coupled to the availability of a sensitive indexing method. In this study, the sensitivity of three BSV detection techniques: rolling circle amplification (RCA), immunocapture PCR (with degenerate and Gold finger primers) and standard PCR was compared. A set of 32 BSD-asymptomatic samples were used to compare the techniques. Analysis of variance (ANOVA) for comparison of the four techniques showed that there were significant differences (P Musa tissues for BSV. This study unveils a more reliable BSV detection method, a need that has remained unaddressed for a long while.
文摘Cyclic polyolefin (COP) is an inexpensive hydrophobic material with low auto-fluorescence, high light transmittance and thermal stability, broad chemical resistance and no non-specific protein binding. Here, the hydrophobic alkane COP was modified to have carbonyl functionalities through oxygen plasma and chemical etching treatments to increase usefulness for chemical and biochemical applications. Then, biotin-hydrazide was used to create biotinylated surfaces that bound streptavidin. A biotinylated target oligonucleotide was subsequently bound to the immobilized biotin-streptavidin and ligation mediated rolling circle amplification-based (L-RCA) SNP detection was demonstrated.
文摘为可视化检测海产品中的活副溶血性弧菌,该研究将叠氮溴化丙锭(propidium monoazide,PMA)染料与跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)技术相结合,以toxR为靶基因,设计并筛选引物,成功建立可视化PMA⁃SRCA检测方法,并评估所建立的方法。研究表明,12株副溶血性弧菌样品呈现阳性,29株非副溶血性弧菌样品呈现阴性,表明该方法特异性良好。该方法灵敏度为3.2×100 CFU/mL,高于可视化PMA⁃环介导等温扩增(3.2×101 CFU/mL)和可视化PMA⁃聚合酶链式反应(3.2×102 CFU/mL)的灵敏度。对76份市售海鲜样品进行检测,该方法的敏感性为100.00%,特异性为92.00%,符合率为97.37%。综上所述,可视化PMA⁃SRCA检测方法可检测活的副溶血性弧菌,具有良好的特异性、较优异的灵敏度。
文摘将生物芯片技术和滚环扩增(rolling circle amplification,RCA)技术结合,建立一种在片RCA检测食源致病性菌新方法.依据待检测致病菌的基因组序列设计捕获探针(capture probe,CP)、检测探针(detect probe,DP)和滚环探针(rolling circle probe,RCP),其中捕获探针、检测探针与待检测致病菌基因组序互补,RCP不含任何待检菌基因组DNA序列,CP的5′末端修饰氨基,将其点样制备成CP微阵列,DP、RCP与待检菌gDNA混合后变性,将其与CP微阵列杂交并连接, RCA扩增反应体系中加入链亲和素进行在片扩增反应,分析检测结果.研究表明,该方法能灵敏、特异性地检测单一和混合靶标分子,以金黄色葡萄球菌为检测对象,其最低检测限可到达50 fg/μL,其线性范围是400 ~50 fg/μL, R 2 达到0.97,证实了该方法的可行性和可靠性,为食源性致病菌检测提供了新的技术方法.
文摘目的:应用滚环扩增(rolling circle amplification,RCA)技术以DNA芯片为载体建立一种对结核分枝杆菌耐药基因单碱基突变的快速检测方法。方法:根据结核分枝杆菌利福平耐药rpoB基因序列,设计针对该基因常见突变位点的锁式探针,及固定于基因芯片上的捕获探针。针对临床结核分枝杆菌样本的基因组DNA,PCR扩增含有rpoB基因常见突变位点的特异性DNA片段。将与突变型特异性互补结合并环化的锁式探针与芯片上固定的捕获探针进行杂交,并运用滚环扩增技术,将含有生物素标记的dUTP掺入扩增产物,最后通过与亲和素标记的纳米金反应,并银染增强显色。同时与临床样本的测序结果比较。结果:通过优化反应条件,能特异性的检测出结核分枝杆菌耐利福平rpoB基因的单碱基突变,通过对临床样本的检测,结果与测序结果一致。结论:该方法结合了DNA芯片和滚环扩增技术,能够快速有效的检测出耐药结核的单碱基突变,具有高特异性、高灵敏度。
基金supported by grants from Jiangsu Higher Education Institution Innovative Research Team for Science and Technology(2021),the Key Technology Program of Suzhou People’s Livelihood Technology Projects(Grant Nos.SKY2021029,SZS2020311)the Open Project of Jiangsu Biobank of Clinical Resources(TC2021B009)the Qing-Lan Project of Jiangsu Province in China(2021,2022).
文摘Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.
文摘该文构建一种基于靶标诱导滚环扩增(rolling circle amplification,RCA)的无标记适配体快速检测赭曲霉毒素A(ochratoxin A,OTA)生物传感器。该生物传感器探针由RCA引物与OTA适配体两部分组成,在OTA存在的环境中,OTA适配体特异性识别靶标,探针结构被打开,RCA引物与环状DNA模板(circular DNA template,CT)结合开启RCA反应,加入核酸染料SYBR Gold产生荧光信号。此生物传感器具有较高的特异性,检测限为6.6×10^(-2)nmol/L,线性检测范围为6.6×10^(-2)~660 nmol/L,可用于具体的分析检测。此生物传感器无需复杂化学修饰且操作简单,在食品安全检测中具有良好的应用前景。
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.
基金financially supported by Hainan Provincial Natural Science Foundation of China(No.822CXTD514)Hainan Province Science and Technology Special Found(No.ZDYF2022SHFZ123)。
文摘Compared with other types of breast cancer,triple-negative breast cancer(TNBC)has the characteristics of a high degree of malignancy and poor prognosis.Early diagnosis of TNBC through biological markers and timely development of effective treatment methods can reduce its mortality.Many Research experiments have confirmed that some specific mi RNA expression profiles in TNBC can used as markers for early diagnosis.However,detecting the expression profiles of multiple groups of miRNAs according to traditional detection methods is complicated and consumes many samples.To address this issue,we developed a method for high-throughput,high-sensitivity quantitative detection of multiple sets of miRNAs(including mi R-16,mi R-21,mi R-92,mi R-199,and mi R-342)specifically expressed in TNBC by rolling circle amplification(RCA)on fluorescence-encoded microspheres.Through the optimization of reaction system conditions,the developed method showed an extensive linear dynamic range and high sensitivity for all five miRNAs with the lowest limit of detection of 2 fmol/L.Meanwhile,this high-throughput detection method also appeared reasonable specificity.Only in the presence of a specific target miRNA,the fluorescence signal on the correspondingly encoded microspheres is significantly increased,while the fluorescence signal on other non-correspondingly encoded microspheres is almost negligible.Furthermore,this process exhibited good recovery and reproducibility in serum.The advantages of this method allow us to more conveniently obtain the expression profiles of multiple groups of TNBC-associated mi RNAs,which is beneficial for the early detection of TNBC.
基金supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20070075003, 20091301120003)the Natural Science Foundation of Hebei Province (B2009000170)
文摘A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.
