Endonuclease-rolling circle amplification-based method for sensitive analysis of DNA-binding protein

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.

Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based Methods in Detection of <i>Banana streak virus</i>in <i>Musa</i>Germplasm

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.

Rolling Circle Amplification on Biotin-Streptavidin Complexes Immobilized to Activated Cyclic Polyolefin Surfaces

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.

High-throughput quantitative detection of triple-negative breast cancer-associated expressed miRNAs by rolling circle amplification on fluorescence-encoded microspheres

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.

Research progress and prospects of nucleic acid isothermal amplification technology

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.

Integration of rolling circle amplification and cationic conjugated polymer for the homogeneous detection of single nucleotide polymorphisms

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%.

跨越式滚环扩增(SRCA)结合金纳米粒子可视化检测食品中的沙门氏菌

沙门氏菌是引起全球人类腹泻病的主要病因,严重危害人畜健康。传统检测方法耗时长,步骤繁琐,因此开展快速简便灵敏的可视化检测方法,具有重要意义。本文设计能与靶序列结合的探针,将其与金纳米粒子(AuNPs)结合。经跨越式滚环等温扩增技术(SRCA)扩增的靶序列,高温变性后成为单链,其与金纳米粒子上的探针结合后,释放出金纳米粒子,在MgSO_(4)的作用下,聚集呈现肉眼可见的蓝色,判定为阳性,阴性为紫色。依此,建立一种简便、快捷、灵敏的可视化检测方法。结果表明:该方法特异性良好,能够区分8株沙门氏菌阳性与8株非沙门氏菌阴性。可视化检测的灵敏度为5.5×10^(0)CFU/mL。将牛奶样品人工污染沙门氏菌,检出限为3.7×10^(0)CFU/mL。在50份实际样品检测中,与GB 4789.4-2016方法进行比较,该方法敏感性为100.00%,特异性为97.87%,符合率达到98.00%。本文建立的SRCA-AuNPs可视化检测方法,具有较高的应用价值,结果肉眼可见,适合于现场可视化检测和基层单位使用。

PMA⁃SRCA可视化检测海产品中的活副溶血性弧菌

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。

Influence of Nucleotide-biased Fluorescence Emissions of SYBR Green Ⅱ on the Result Consistence of Rolling Circle Amplification

The fluorescence dye SYBR Green Ⅱ(SG Ⅱ)has been frequently used in rolling circle amplification(RCA)based analyses of nucleic acids.However,a good amount of inconsistencies have been reported in regards the quality and reproducibility of RCA reactions.To properly examine this experimental issue,here we utilized a series of synthetic oligonucleotides and circular templates to investigate the impact of SG Ⅱ in RCA reactions.The results indicate that SG Ⅱ enables a strong fluorescence signal only when complexing with guanosine(G)residue.In RCA reactions,long single-stranded RCA products,enriched with G residues,result in higher fluorescence emission when compared with the addition of other nucleotide residues.These results suggest that the nucleotide composition of the reaction can affect the amplification results and,eventually,can lead to inconsistent fluorescence of the RCA products.This work indicates that particular attention should be given when circular templates are designed for the quantitative analysis of nucleic acids,to further allow the signal reproducibility of RCA-based experiments.

Self-assembly of DNA Origami Using Rolling Circle Amplification Based DNA Nanoribbons

During the development of structural DNA nanotechnology,the emerging of scaffolded DNA origami is marvelous.It utilizes DNA double helix inherent specificity of Watson-Crick base pairing and structural features to create self-assembling structures at the nanometer scale exhibiting the addressable character.However,the assembly of DNA origami is disorderly and unpredictable.Herein,we present a novel strategy to assemble the DNA origami using rolling circle amplification based DNA nanoribbons as the linkers.Firstly,long single-stranded DNA from Rolling Circle Amplification is annealed with several staples to form kinds of DNA nanoribbons with overhangs.Subsequently,the rectangle origami is formed with overhanged staple strands at any edge that would hybridize with the DNA nanoribbons.By mixing them up,we illustrate the one-dimensional even two-dimensional assembly of DNA origami with good orientation.

Dual Rolling Circle Amplification- Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Exosomes secreted by tumor cells carry abundant molecular biomarkers that reflect the status of their originating cells.These tumor-derived exosomes(TDEs)have emerged as attractive diagnostic targets.However,the identification and characterization of highly heterogeneous TDEs remain practically challenging.Here,we report a dual rolling circle amplification(DRCA)-assisted approach for the selective encapsulation of single TDEs for fluorescence microscopic and flow cytometric analysis.TDEs have been targeted by aptamers that recognized their surface tumor marker and exosomal marker CD63,following DRCA that produced entangling polymeric DNA chains,resulting in facile particle enlargement that allows single-particle fluorescence profiling of exosome heterogeneity.We have demonstrated the use of a dual-marker positive ratio for exosome differentiation and applied division and multiplication operations for normalized andmagnified marker heterogeneity analysis.We further applied this assay to distinguish lung adenocarcinoma and pulmonary nodule patients and found an accuracy of 90%.We anticipate promising transformations of this straightforward assay into clinically implantable diagnostic methods.

食源性致病菌生物芯片-RCA检测新方法

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.

RCA技术检测甲型H1N1流感病毒耐药基因单碱基突变的应用研究

目的建立一种检测甲型H1N1流感病毒耐药基因单碱基突变的滚环扩增技术(rolling cycle amplification,RCA)。方法以甲型H1N1流感病毒耐药基因M2基因和NA基因为研究对象,设计检测该基因突变位点的环化探针,环化探针通过与发生单碱基突变的基因特异性结合并被连接成闭合环状,进行滚环扩增后从而特异性地检测单碱基突变。讨论用于该检测的RCA技术的合适反应条件,通过对人工合成的野生型和突变型靶序列检测,确定该方法的特异性和灵敏度。最后通过对临床标本的检测及与测序结果比对,验证该方法的准确性。结果检测单碱基突变需要严格的反应条件,采用热循环连接法和提高连接温度(65℃),保证了检测特异性。通过对含有不同浓度突变靶序列标本的检测确定了该方法能检测出最低1%含量的突变株。RCA技术检测结果与测序方法结果一致。结论成功建立检测甲型H1N1流感病毒耐药基因单碱基突变的RCA技术。

RCA技术联合DNA芯片检测结核分枝杆菌rpoB基因突变

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。

基于PLP-LDR-HRCA策略进行微量DNA分析——rs17750303位点分型

增强PCR和全基因组扩增是当前微量DNA分析的主要策略,但是,由于DNA模板量过少,受随机效应影响显著,往往不能得到可靠的DNA分型结果.本文提出一种新的检验策略:PLP-LDR-HRCA,尝试微量DNA检材的SNPs分型研究.选择rs17750303位点,并设计等位基因特异性锁式探针,采用连接酶检测反应来识别等位基因,而后采用超分支滚环扩增反应来放大检测信号.结果表明,PLP-LDR-HRCA反应特异性好,灵敏度高,能够直接鉴别微量基因组DNA模板中待测SNP位点,rs17750303纯合型样品(AA型或CC型)和杂合型样品(AC型)准确分型所需最少模板量分别为20pg和30pg.对于增强PCR和全基因组扩增技术不能有效检验的微量检材,PLP-LDR-PCR策略独具优势,可能具有较大的开发价值.

基于G-四链体的PCR-RCA双重扩增技术检测沙门氏菌

将聚合酶链式反应(polymerase chain reaction,PCR)与滚环扩增技术(rolling circle amplification,RCA)联用,并把G-四链体互补序列嵌入到RCA扩增所需的哑铃环状模板上,通过PCR和RCA的双重扩增及特异性结合G-四链体的硫黄素T荧光信号增强的作用,达到基于G-四链体的PCR-RCA技术检测沙门氏菌(Salmonella)的目的。在优化的检测体系下,确定了沙门氏菌基因组DNA浓度对数与486 nm波长处的荧光信号强度具有良好的线性关系,回归方程为y=2.101x+2.8723(R^(2)=0.9925),线性范围为17 fg/μL~1.7 ng/μL。根据实验的特异性分析,表明此方法适用于沙门氏菌属的检测,对人工污染沙门氏菌牛奶样品进行检测,检出限为4.28 CFU/mL。该方法具有特异性强、灵敏度高、检出限低等优点,为实现食源性致病菌的快速检测提供新的方法。

SNP与基因检测及表达中的RCA技术

滚环扩增(rollingcircleamplification,RCA)技术是一种新的分子生物学检测方法。该方法不仅可以在体外等温条件下对核酸进行高度特异性的检测,而且还可通过线性或指数扩增来进行信号级联放大,其灵敏度能达到1个拷贝的核酸分子,因此,可用于痕量分子的检测。目前,滚环扩增技术广泛应用于全基因组DNA检测、核酸测序、单核苷酸多态性、DNA芯片及蛋白质芯片分析等领域。

PMA-RF-SRCA检测乳中单增李斯特氏菌活菌

本研究建立了一种叠氮溴化丙锭(PMA)与实时荧光跨越式滚环等温扩增(RF-SRCA)相结合的方法,可以高效灵敏检测乳中活的单增李斯特氏菌。以hlyA基因为靶点,设计并筛选出特异性引物,通过对PMA的工作浓度、暗孵育时间和光反应时间进行优化,确定了最佳的处理条件。此外,对该方法的特异性、灵敏度及检出限进行了分析。结果表明,该方法引物特异性良好,6株单增李斯特氏菌结果均为阳性,12株非单增李斯特氏菌结果均为阴性;当PMA工作浓度为30μmol/L、暗孵育10 min、光反应15 min时,所建立的方法灵敏度为3.9 CFU/mL,人工污染乳制品的检出限为1.56×10 CFU/mL。综上所述,本研究所建立的PMA-RF-SRCA方法特异性强,灵敏度高,为检测食品中活的单增李斯特氏菌提供了新的思路。

实时荧光跨越式滚环等温扩增技术检测蜂蜜掺伪大米糖浆

将跨越式滚环等温扩增技术(SRCA)与荧光技术相结合,建立一种实时荧光SRCA技术检测蜂蜜掺伪大米糖浆的方法。对DNA提取方案进行评估,以大米的特异性基因(PDL、SPS、rbcL、GOS9)为靶序列设计引物,筛选适宜的引物,优化扩增反应条件结合荧光技术建立检测蜂蜜掺伪大米糖浆的方法,并对该方法进行评价。结果表明,建立的实时荧光SRCA方法检测大米DNA的灵敏度为8.45×10~1fg/μL,经特异性评价证实其特异性良好,在人工模拟掺伪检测中建立掺伪比例的对数与Ct值的线性关系,线性方程为y=6.618x+7.651(R~2=0.993),可准确检出蜂蜜中低至1%的大米糖浆成分。该方法灵敏度高,检出限低,能够快速、准确检测蜂蜜掺伪大米糖浆,为蜂蜜掺伪的快速检测提供了新思路。

重症肌无力患者血浆染色体外环状DNA的分子特征

目的对重症肌无力(myasthenia gravis,MG)患者血浆染色体外环状DNA(extrachromosomal circular DNA,eccDNA)进行全长测序,分析eccDNA的分子特征及潜在功能,初步探索eccDNA在MG发病过程中的作用机制。方法收集2例MG患者及2例性别、年龄与之匹配的健康人血浆样本,基于滚环扩增和纳米孔测序全新技术平台,对血浆eccDNA进行全长测序,分析比较MG患者与健康人血浆eccDNA的长度分布、染色体来源、基因组元件分布及eccDNA相关差异基因功能富集情况。结果在MG患者和健康对照者中,长度为250~500 bp的eccDNA均分布最多,且MG患者在150~300 bp之间eccDNA呈现另一分布高峰,对照组则在此区间的eccDNA丰度极低。健康对照组eccDNA在1号染色体上分布最多,而MG患者组eccDNA在2号染色体上分布最多;MG患者eccDNA来源基因组元件在内含子、远端基因间区占比均高于健康对照者,而外显子区占比均低于健康对照者。相较于健康对照组,MG患者组eccDNA差异基因富集的通路多与氯离子通道活性、氯离子跨膜转运、钙离子结合及细胞信号传导有关。结论MG患者与健康人血浆eccDNA的分子特征(大小分布、染色体来源、基因组元件分布、eccDNA基因功能富集)存在差异,提示eccDNA可能通过基因表达调控、细胞信号传导、神经突触发育及免疫功能调节等潜在功能影响MG的发生发展,eccDNA可能成为MG早期诊断和疗效监测的新型生物标志物。