滚环扩增技术在电化学生物传感器中的应用

在电化学生物传感器的设计中,信号放大是实验环节中的重要步骤,特别是对靶标进行灵敏度分析时更是不可或缺。滚环扩增（rolling circle amplification,RCA）能够在短时间内得到大量产物,并在电极表面进行扩增或孵育,然后通过一定的设计使电化学信号被快速放大。RCA技术具有高度的灵敏性和特异性,电化学生物传感器则可提供实时、快速、低成本的检测。为了更好的了解RCA,介绍了RCA环化的基本原理、RCA种类,重点总结了RCA与电化学生物传感器结合的不同技术类型及应用,并对未来相关研究领域的发展趋势进行了展望,旨在为RCA技术在电化学生物传感器中的进一步发展和应用提供参考。     

滚环扩增信号放大技术在生物检测中应用的研究进展

滚环扩增(Rolling circle amplification,RCA)是一种快速、灵敏且恒温的单链DNA(Single-stranded DNA,ssDNA)扩增技术,与染色或探针联用可实现检测信号的放大,在生物检测等方面得到广泛的应用。文中对RCA的构建方法进行了简介,综述了近几年其在致病菌、核酸肿瘤标记物、蛋白质、生物小分子和病毒等检测中的研究进展,并对其未来的发展趋势进行了展望。     

滚环DNA扩增的原理、应用和展望

滚环DNA扩增 (rollingcircleDNAamplification ,RCA)是一种等温信号扩增方法 ,其线性扩增倍数为 1 0 ⁵,指数化扩增能力大于 10⁹,产生的扩增产物连接在固相支持物 (如玻片、微孔板等 )表面的DNA引物或抗体上。RCA是一种适合在芯片上 (on chip)进行信号扩增的新技术 ,它既能提供研究分析的敏感性和特异性 ,又能保持立体分析的多元性。RCA亦是一种痕量的分子检测方法 ,可用于极其微量的生物大分子和生物标志的检测与研究     

利用滚环扩增鉴别同源家族miRNAs

滚环扩增（rolling circle amplification, RCA）是一种基于病毒DNA复制而发明的新技术。近些年,RCA技术已经被广泛应用于微小核糖核酸（micro ribonucleic acid, miRNA）的检测。在miRNA检测研究领域中,鉴别高度同源的家族miRNAs成为该研究领域的瓶颈。本研究引入新型的RCA技术来增加鉴别的灵敏度和特异性,进一步提高家族miRNA鉴别的灵敏度,滚环扩增的程度用相对荧光强度来表示。研究结果显示,T4 RNA连接酶2可在RCA的环化过程中实现最大的环化效率,从而提高RCA的检测特异性。本文利用优化的RCA技术,实现对let 7高度同源的家族miRNAs高灵敏度的鉴别,灵敏度可达5 fmol。let 7a的滚环探针对Let 7a这一miRNA扩增后的相对荧光强度为1 550,而对其他的家族miRNA相对荧光强度仅为260。其他的家族miRNA探针在鉴别时相对荧光强度也显示了较大的差异。而依靠传统的RT-qPCR方法的鉴别灵敏度是4 pmol,与本研究相比,灵敏度低了近1 000倍。本研究的结果表明,利用RCA技术鉴别高度同源性miRNAs是高效灵敏的,此前未见相关研究的报道。RCA技术可能被应用于miRNA高灵敏度检测和鉴别的相关研究中。     

利用滚环扩增鉴别同源家族miRNAs

滚环扩增（rolling circle amplification, RCA）是一种基于病毒DNA复制而发明的新技术。近些年,RCA技术已经被广泛应用于微小核糖核酸（micro ribonucleic acid, miRNA）的检测。在miRNA检测研究领域中,鉴别高度同源的家族miRNAs成为该研究领域的瓶颈。本研究引入新型的RCA技术来增加鉴别的灵敏度和特异性,进一步提高家族miRNA鉴别的灵敏度,滚环扩增的程度用相对荧光强度来表示。研究结果显示,T4 RNA连接酶2可在RCA的环化过程中实现最大的环化效率,从而提高RCA的检测特异性。本文利用优化的RCA技术,实现对let 7高度同源的家族miRNAs高灵敏度的鉴别,灵敏度可达5 fmol。let 7a的滚环探针对Let 7a这一miRNA扩增后的相对荧光强度为1 550,而对其他的家族miRNA相对荧光强度仅为260。其他的家族miRNA探针在鉴别时相对荧光强度也显示了较大的差异。而依靠传统的RT-qPCR方法的鉴别灵敏度是4 pmol,与本研究相比,灵敏度低了近1 000倍。本研究的结果表明,利用RCA技术鉴别高度同源性miRNAs是高效灵敏的,此前未见相关研究的报道。RCA技术可能被应用于miRNA高灵敏度检测和鉴别的相关研究中。     

滚环扩增技术及其在生物分子诊断中的应用

滚环扩增技术(RCA)是近年来发展起来的一种新型的核酸扩增技术.该技术是基于连接酶连接、引物延伸、与链置换扩增反应的一种等温核酸扩增方法.在恒温的条件下,可以产生大量的与环型探针互补的重复序列.与传统的核酸扩增方法相比,它具有扩增条件简单,特异性高,能在恒温条件下进行等特点.滚环扩增技术结合荧光、电化学、电化学发光等检...     

柑桔溃疡病菌滚环扩增检测体系的建立

根据柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,Xac)独有的蛋白基因序列和锁式探针公共连接序列分别设计特异性的锁式探针及其扩增引物,优化系列反应条件,建立了特异性的柑桔溃疡病菌滚环扩增体系.初步检测结果表明该体系能够特异性地检出Xac的菌体细胞及其DNA,而检测不出供试的其它植物病原细菌和柑桔叶面常见的多种附生细菌;对Xac靶片段克隆质粒DNA的检测灵敏度为10 2 copy/μL,对Xac菌悬液的检测灵敏度为20 cfu/μL,比常规PCR的检测灵敏度稍高.用滚环扩增技术和常规PCR技术对田间采集的实际样品进行了检测,两种方法的检测结果没有显著差异(P＞0.01).由于锁式探针的公共连接序列对扩增的条件要求一致,本体系的建立可以为植物病原微生物多靶标检测和病害检疫检验提供新的技术支撑.     

一种DNA扩增的新技术： 利用热稳定的Bst DNA聚合酶驱动跨越式滚环等温扩增反应

Bst DNA聚合酶具有热稳定性、链置换活性及聚合酶活性,在体外DNA等温扩增反应中起重要作用. 本文利用Bst DNA聚合酶的5′→3′聚合酶、核苷酸（末端）转移酶及链置换酶活性发展了一种新的体外环式DNA扩增技术跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)．在SRCA反应中,Bst DNA聚合酶以上游引物P1为模板合成其互补链RcP1,并和P1形成双链DNA|之后,Bst DNA聚合酶用其核苷酸转移酶活性在其P1的3′末端沿5′→3′方向随机掺入脱氧核糖核苷酸聚合形成寡聚核苷酸（dNMP)m序列,即DNA的合成反应跨越了RcP1 与下游引物P2之间的缺口|然后,以下游引物P2为模板形成互补序列（RcP2）;接着,Bst DNA聚合酶继续将脱氧核糖核苷酸随机添加到RcP2的3′末端,形成（dNMP)n序列．继而,Bst DNA聚合酶以RcP1为模板,继续催化聚合反应合成互补新链,并通过其链置换酶活性替换P1|如此往复,形成［P1-(dNMP)m-RcP2-(dNMP)n …］序列．本文通过电泳、酶切、测序等方法对扩增产物进行分析,演绎出上述扩增过程,并就工作原理进行了讨论．该反应可能对开发等温扩增技术检测微生物有一定助益,也为解释环介导等温扩增技术中假阳性反应和滚环等温扩增反应中的背景信号提供了线索．     

基于滚环扩增的鸭乙型肝炎病毒共价闭合环状DNA检测方法的建立

滚环扩增(RCA)是新近发展起来的一种能特异性扩增环形DNA的实验技术,自2008年以来被广泛用于HBV基因全长扩增及共价闭合环状DNA(cccDNA)耐药突变分析等研究。为了便于鸭乙型肝炎病毒(DHBV)cccDNA的分析,本研究建立了基于RCA的DHBV cccDNA的检测方法。通过针对DHBV高度保守序列设计的4对RCA硫化修饰引物,以血清DHBV DNA为阴性对照,从肝组织DHBV DNA标本中扩增得到DHBV cccDNA。然后用跨缺口引物扩增RCA产物测序替代限制性内切酶切分析进行DHBV cccDNA鉴定。应用该方法检测39份携带DHBV麻鸭肝组织与血清标本结果显示:全部肝组织标本均检出DHBV cccDNA,而全部血清标本则均无DHBV cccDNA检出,表明本研究建立的基于RCA的DHBV cccDNA检测法具有良好的特异性和灵敏性。该方法的建立为应用鸭乙型肝炎病毒动物模型研究cccDNA在病毒致病机制中的作用以及评价抗病毒疗效奠定了实验基础。     

核酸恒温扩增技术研究进展

核酸恒温扩增技术在生命科学研究及相关诸多领域已经得到了广泛应用。我们对核酸恒温扩增技术的最新进展作一简要综述,包括环介导恒温扩增、链替代扩增、依赖核酸序列的扩增、滚环扩增、切口酶核酸恒温扩增、依赖解旋酶的恒温扩增、转录依赖的扩增、杂交捕获法、转录介导的扩增等的原理、优缺点及应用。     

Target-cycling synchronized rolling circle amplification strategy for biosensing Helicobacter pylori DNA

Helicobacter pylori is closely linked to many gastric diseases such as gastric ulcers and duodenal ulcers. Therefore, biosensing H. pylori has attracted wide attention from both scientists and clinicians. Here, we proposed an electrochemiluminescence (ECL)-based platform that could sensitively detect H. pylori DNA. In this platform, a novel target-cycling synchronized rolling circle amplification was used for signal amplification. Silver nanoclusters (Ag NCs) were synthesized on the circle DNA products, embedding them with the ability to catalyze the electrochemical reduction of K₂S₂O₈, in turn resulting in rapid consumption of the ECL co-reactant near the working electrode, and leading to a decrease in the ECL emission intensity. In addition to its excellent stability and selectivity, the proposed strategy had a low detection limit of 10 pM, an indication that it can be beneficially applied to test biosamples. Furthermore, a biosensing chip was designed to improve the throughput and shed new light on large-scale clinical biosensing applications.     

Hyperbranched rolling circle amplification as a novel method for rapid and sensitive detection of Amphidinium carterae

High quality of coastal water is critical to marine ecosystems, marine fisheries, public health, and aquatic environment. Specially, bio-toxin derived from toxic microalgae is currently threatening many coastal countries. Therefore, development of rapid and sensitive methods for the detection of toxin-producing microalgae is necessary for warning of water quality. In this paper, we established a novel method for rapid and sensitive detection of Amphidinium carterae by hyperbranched rolling circle amplification (HRCA). The partial large subunit rDNA (LSU D1–D2) of A. carterae was sequenced to design species-specific padlock probe (PLP). The PLP-coupled with two amplification primers were employed for HRCA. The optimized HRCA conditions were as follows: padlock concentration, 20 pM; ligation temperature, 65 °C; ligation time, 15 min; amplification temperature, 61 °C; and amplification time, 15 min. The developed HRCA was confirmed to be specific for A. carterae by tests with other algae. The sensitivity of HRCA was 100-fold higher than regular PCR, exhibiting a detection limit of 1 fg/μL representing 283 copies for the recombinant plasmid containing the target LSU D1–D2, and 1 cell for target species. Finally, a simplified protocol was applied to the simulated field and environmental materials, and exhibited a good performance. The whole detection could be completed within 1.5 h, displaying a repeated detection limit of 1 cell. The positive HRCA results could be visualized through coloration reaction by adding the fluorescent dye SYBR Green I to the amplification products. The HRCA provides a useful tool to quickly screen large sample sets for A. carterae, as well as other toxic species.     

Homogeneous and label-free fluorescence detection of single-nucleotide polymorphism using target-primed branched rolling circle amplification

We present a simple, sensitive, and cost-effective fluorescent assay of single-nucleotide polymorphism (SNP) with target-primed branched rolling circle amplification (TPBRCA). Designed padlock probe is circularized after perfect hybridization to mutant DNA. Then rolling circle amplification (RCA) reaction can be initiated from the mutant DNA that acts as primer and generates a long tandem single-stranded DNA (ssDNA) product. At the same time, the introduction of a reverse primer complementary to the target-primed RCA products leads to the branched RCA and eventually generates the various lengths of ssDNA and double-stranded DNA products, which are sensitively detected using SYBR Green I (SG) fluorescence dye. In contrast, the wild DNA contains a single mismatched base with the padlock probe and primes only a limited extension with the unligated padlock probe, generating weak background fluorescence with the addition of SG. Due to the excellent specificity and powerful amplification of TPBRCA reaction, the mutant DNA was distinctively differentiated from the wild DNA in a homogeneous and label-free manner. The assay is sensitive and specific enough to detect 5-amol (8.6-fM) mutant DNA strands. It was possible to accurately determine the mutant allele frequency as low as 1.0%.     

Establishment and application of hyperbranched rolling circle amplification coupled with lateral flow dipstick for the sensitive detection of Karenia mikimotoi

The dinoflagellate Karenia mikimotoi is a noxious and harmful algal bloom (HAB)-forming microalga. Establishing a rapid, accurate, and sensitive method of detecting this harmful alga is necessary to provide warnings of imminent HABs through field monitoring. Here, an isothermal amplification technique combined with a rapid analytical method for nucleic acid-based amplified products, i.e., hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD), hereafter denoted as HRCA-LFD, was established to detect K. mikimotoi. The HRCA-LFD assay relied on a padlock probe (PLP) targeting DNA template and an LFD probe targeting PLP. The sequenced internal transcribed spacer of K. mikimotoi through molecular cloning was used as the target of PLP. The optimized HRCA conditions was determined to be as follows: PLP concentration, 20 pM; ligation temperature, 65 °C; ligation time, 10 min; amplification temperature, 61 °C; and amplification time, 30 min. The developed HRCA-LFD assay was specific for K. mikimotoi, displaying no cross-reactivity with other common microalgae. Sensitivity-comparison tests indicated that HRCA-LFD assay was 100-fold more sensitive than PCR, with a detection limit of 0.1 cell mL⁻¹ when used to analyze spiked field samples. The analysis with field samples also indicated that HRCA-LFD assay was suitable for samples with a target cell density range of 1–1000 cells mL⁻¹. All of these results suggested that HRCA-LFD assay is an alternative method for the sensitive and reliable detection of K. mikimotoi from marine water samples.     

Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases

Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2′-OMeRNA modification, which might be due to the size of the C2′-modified moieties. 2′-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction.     

Characterization of putative circular plasmids in sponge‐associated bacterial communities using a selective multiply‐primed rolling circle amplification

Plasmid transfers among bacterial populations can directly influence the ecological adaptation of these populations and their interactions with host species and environment. In this study, we developed a selective multiply‐primed rolling circle amplification (smRCA) approach to enrich and characterize circular plasmid DNA from sponge microbial symbionts via high‐throughput sequencing (HTS). DNA (plasmid and total community DNA) obtained from sponge (Cinachyrella sp.) samples and a bacterial symbiont (Vibrio sp. CyArs1) isolated from the same sponge species (carrying unknown plasmids) were used to develop and validate our methodology. The smRCA was performed during 16 hr with 141 plasmid‐specific primers covering all known circular plasmid groups. The amplified products were purified and subjected to a reamplification with random hexamer primers (2 hr) and then sequenced using Illumina MiSeq. The developed method resulted in the successful amplification and characterization of the sponge plasmidome and allowed us to detect plasmids associated with the bacterial symbiont Vibrio sp. CyArs1 in the sponge host. In addition to this, a large number of small (<2 kbp) and cryptic plasmids were also amplified in sponge samples. Functional analysis identified proteins involved in the control of plasmid partitioning, maintenance and replication. However, most plasmids contained unknown genes, which could potentially serve as a resource of unknown genetic information and novel replication systems. Overall, our results indicate that the smRCA‐HTS approach developed here was able to selectively enrich and characterize plasmids from bacterial isolates and sponge host microbial communities, including plasmids larger than 20 kbp.     

Detection and identification of opportunistic Exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers

Deep infections by melanized fungi deserve special attention because of a potentially fatal, cerebral or disseminated course of disease in otherwise healthy patients. Timely diagnostics are a major problem with these infections. Rolling circle amplification (RCA) is a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of microorganisms. RCA-based diagnostics are characterized by good reproducibility, with few amplification errors compared to PCR. The method is applied here to species of Exophiala known to cause systemic infections in humans. The ITS rDNA region of five Exophiala species (E. dermatitidis, E. oligosperma, E. spinifera, E. xenobiotica, and E. jeanselmei) was sequenced and aligned in view of designing specific padlock probes to be used for the detection of single nucleotide polymorphisms (SNPs) of the Exophiala species concerned. The assay proved to successfully amplify DNA of the target fungi at the level of species; while no cross-reactivity was observed. Amplification products were visualized on 1% agarose gels to verify the specificity of probe-template binding. Amounts of reagents were minimized to avoid the generation of false positive results. The sensitivity of RCA may help to improve early diagnostics of these difficult to diagnose infections.     

Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500×. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets.