HRCA技术检测中华鳖虹彩病毒

[目的]中华鳖虹彩病毒(STIV)是甲鱼重要的病毒性病原之一,建立特异性好、灵敏度高的中华鳖虹彩病毒超分支滚环扩增体系(HRCA),对STIV进行快速、准确地检测.[方法]根据中华鳖虹彩病毒(STIV)独有的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,对HRCA的系列反应条件进行优化,验证该方法的特异性和灵敏度,并用该方法对感染STIV显症和未显症的甲鱼组织进行检测.[结果]10 nmol/L探针经T4 DNA连接酶作用20 min环化,Bst DNA聚合酶大片段扩增20 min即可获得很好的检测效果.特异性试验表明,在多种待检病毒样中,HRCA能特异地检测出STIV,同时HRCA具有极高的灵敏度,能检测出的最低模板量为101拷贝,而常规PCR的检测下限为103拷贝,对显症和尚未显症的感染早期甲鱼组织的检测结果均为阳性.[结论]建立的中华鳖虹彩病毒HRCA检测体系具有灵敏、快速、简便等特点,能从组织样品中准确地检测出STIV,可以用于该类疾病的早期诊断,具有较好的推广前景.     

基于锁式探针技术快速检测玉米细菌性枯萎病菌和玉米内州萎蔫病菌

目的:建立一种检测玉米细菌性枯萎病菌和玉米内州萎蔫病菌的方法,为同时检测这2种检疫性细菌提供技术手段。方法:基于靶标序列设计2种检疫性细菌的锁式探针,与靶标菌进行连接消化反应,然后采用通用引物进行滚环扩增,其产物与偶联上对应捕获探针的微球进行杂交,最后通过液相悬浮芯片二重检测。结果:该检测方法能够有效地检测2种检疫性细菌,其检测阈值为103CFU/m L,具有良好的可重复性。结论:建立了一种快速、灵敏的玉米细菌性枯萎病菌和玉米内州萎蔫病菌的二重检测方法。     

一种核糖体展示单链抗体文库的构建与鉴定

用柑桔溃疡病致病菌Xanthomonas axonopodis pv. citri（Xac）全菌免疫小鼠,提取小鼠脾细胞mRNA,RT-PCR扩增小鼠抗体重链可变区（VH）和轻链可变区（VL）,采用linker (Gly4Ser)3连接VH和VL,构建用于核糖体展示方法筛选阳性单链抗体（scFvs）的文库。通过将scFv文库DNA转化大肠杆菌JM109,随机挑取克隆子测序以分析单链抗体文库的多样性。结果显示,用柑桔溃疡病菌免疫后的小鼠抗血清效价为2500倍左右,扩增的VH大小为350bp左右,VL的大小为650bp左右,linker连接后的单链抗体文库DNA大小为1200bp左右。测序结果显示,单链抗体文库多样性好。以Xac为靶,筛选到了抗Xac的特异性抗体,说明该抗体库可用于柑桔溃疡病菌单链抗体的筛选。     

滚环复制技术的建立及在RNA病毒基因检测中的初步应用

滚环复制是噬菌体繁殖所采取的一种基因复制方式,这种方式可使单链的环形分子在聚合酶和引物的作用下进行体外自我扩增。本文中用可特异性连接环化的寡核苷酸链作为探针,分别进行了1份细胞培养的禽流感病毒H5N1亚型样品、1份细胞培养的SARS病毒样品和4份丙型肝炎病毒阳性血清样品的检测。检测原理是探针与靶序列杂交后便可在T4DNA连接酶的作用下形成滚环复制中的环化单链分子,该分子在同温下可被特异性引物滚动复制和支链扩增。本文还利用按禽流感病毒NA1基因区序列合成的模拟DNA分子对该检测方法的灵敏度进行了测试。结果显示：利用固相RCA技术成功检测到三种RNA病毒的基因,该方法的灵敏度可达到能检测10^3拷贝模式DNA分子的水平。与传统的PCR方法敏感性的比较尚待进一步研究。     

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.     

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。     

滚环扩增技术的原理及其应用的研究

滚环扩增是近年来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,RCA技术在全基因组扩增、单核苷酸多态性、DNA芯片、蛋白质芯片等方面检测中具有很大的应用价值和潜力。     

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。     

四种黄单胞菌的基因芯片检测方法的建立

结合双重PCR和基因芯片技术同时检测和鉴定我国检疫性细菌,包括水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)、水稻细菌性条斑病菌(X.oryzae pv.oryzicola,Xooc)、柑桔溃疡病菌(X.axonopodis pv.citri,Xac)以及严重危害十字花科作物的甘蓝黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)。以铁载体受体(Putative siderophore receptor)基因序列和RNA多聚酶西格玛因子(RNA polymerase sigma factor,rpoD)基因序列为靶标,设计引物和特异性探针能够同时检测这4种重要的病原菌。对17个细菌菌株进行芯片检测,仅4种靶标菌得到阳性结果,证明此方法具有很高的特异性。4种致病菌基因组DNA的检测灵敏度约为3 pg。检测结果表明,建立的基因芯片检测方法特异性强,能实现上述4种黄单胞菌的准确检测和鉴定,具有良好的应用前景。     

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。     

HRCA技术在转基因植物检测中的应用

超分支滚环扩增技术（Hyperbranched rolling cycle amplification, HRCA）在近几年中逐渐引起人们的注意,并越来越多的用于基础研究和实际检测中。在本文中我们对该技术在转基因植物检测中的应用情况作了较全面的探索;根据4种转基因植物中常用的外源基因或DNA片段设计了4条锁式探针（Padlock probe）,利用质粒pKK2328中的一段序列作为锁式探针中的共同连接部分,并根据该共同的连接部分序列设计一对通用的HRCA引物;利用同位素标记的锁式探针对HRCA反应中的连接一步的特异性研究表明,只有当锁式探针和相应的检测靶DNA同时存在于连接体系中时,锁式探针才能被有效地进行连接,从线性分子变为环型分子,在没有相应靶DNA存在时锁式探针仅以线性形式存在;连接时间的研究表明,如果所检测的靶DNA是质粒或较短的DNA片段时,较短的连接时间（5～10min）就可以取得理想的最终检测效果,如果检测的靶DNA是复杂的植物基因组DNA时,连接时间需要较大程度的延长（30～60min）才能取得理想的最终检测结果;HRCA的反应时间研究表明,较长的反应时间可以明显增加最终产物的量;对Bst DNA聚合酶大片段酶用量的研究表明,在其它条件不变的情况下酶的用量可以在较大的范围内变化（0.5u～4u）而不影响最终检测结果;在上述研究的基础上,对转基因烟草进行实际检测,取得了与预期一致的理想结果。为了提高检测效率,仿效复合式PCR（Multiplex PCR,MPCR）的原理采用复合式HRCA（Multiplex HRCA, MHRCA）方法对转基因烟草进行检测,并利用反向点杂交进行结果分析,取得同预期完全一致的结果。我们的研究表明HRCA方法完全可以用于转基因植物的检测,而且其使用比MPCR技术更方便,效率更高。     

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%.     

Primers based on the rpf gene region provide improved detection of Xanthomonas axonopodis pv. citri in naturally and artificially infected citrus plants

AIMS: To have a PCR-based detection method for Xanthomonas axonopodis pv. citri (Xac) using primers designed in a specific region of its genome. METHODS AND RESULTS: A Xac-specific region was identified inside the rpf gene cluster of strain IAPAR 306 in an analysis of its complete genomic sequence. Two primers were designed, Xac01 and Xac02, which, when used in a standard PCR assay, direct the amplification of a 581 bp fragment from DNA of strains belonging to Xac from different regions around the world including unusual American and Asian strains. This product was not observed when DNA from strains of the closely related X. a. aurantifolli and X. a. citrumelo were used as templates. Extracts prepared from 28 xanthomonads of other species, and epiphytic bacteria isolated from citrus also failed to produce products with these primers. Amplification was obtained from cells grown in vitro, from extracts of both fresh and dried citrus canker lesions and from washes of inoculated but asymptomatic leaf surfaces. In sensitivity tests, this PCR technique detected as few as 100 cells. CONCLUSIONS: Primers Xac01 and Xac02 provide specific and sensitive detection of Xac in all citrus tissues where the pathogen is found. SIGNIFICANCE AND IMPACT OF THE STUDY: This PCR-based diagnostic test is suitable for monitoring asymptomatic plants in areas where the bacteria is endemic, in plant quarantine and regulatory situations, and also for obtaining an accurate diagnosis in a very short time. These are important characteristics for any assay to be used for the management of citrus canker disease.     

Diagnosis of Xanthomonas axonopodis pv. citri, causal agent of citrus canker, in commercial fruits by isolation and PCR-based methods

AIMS: To show the results of the detection of an EU quarantine organism, Xanthomonas axonopodis pv. citri (Xac), in citrus fruits imported from countries where this bacterium is present, using an integrated approach that includes isolation, pathogenicity assays and molecular techniques. METHODS AND RESULTS: Citrus fruits with canker-like symptoms, exported to Spain from South American countries were analysed by several methods. Bacterial isolation, three conventional polymerase chain reaction (PCR) protocols, and real-time PCR with SYBR Green or a TaqMan probe, were compared. Canker-like lesions were disrupted in PBS buffer, and the extract used for bacterial isolation and DNA extraction followed by PCR amplification. Canker lesions, identified by PCR, showed viable bacteria in eleven of fifteen fruit samples. In 16 out of 130 lesions analysed from these samples, Xac was isolated, and pathogenicity on grapefruit leaves confirmed. By real-time PCR, using SYBR green or a Taqman probe, Xac was detected in 58 and 80 lesions respectively. By conventional PCR the bacterium was detected in 39-52 lesions depending on the protocol employed. CONCLUSIONS: An integrated approach for reliable detection of Xac in lesions of fruit samples, employing several techniques and with real-time PCR using a TaqMan probe as the fastest and most sensitive screening method, has been established and validated and is proposed as a useful tool for the analysis of Xac on fresh fruits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work faces up to the real threat of the importation of citrus fruits that can harbour quarantine bacteria and will be useful in diagnostic laboratories for the analysis of commercial fresh fruits from countries where citrus canker is present.     

Sensitive and isothermal electrochemiluminescence gene-sensing of Listeria monocytogenes with hyperbranching rolling circle amplification technology

Listeria monocytogenes (L. monocytogenes) is one of the most problematic human pathogens, as it is mainly transmitted through the food chain and cause listeriosis. Thus, specific and sensitive detection of L. monocytogenes is required to ensure food safety. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with magnetic beads based electrochemiluminescence (ECL) to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. At first, a linear padlock probe was designed to target a specific sequence in the hly gene which is specific to L. monocytogenes and then ligated by Taq DNA ligase. After ligation and digestion, further amplification by HRCA with a biotiny labeled primer and a tris (bipyridine) ruthenium (TBR) labeled primer was performed. The resulting HRCA products were then captured onto streptavidin-coated paramagnetic beads and were analyzed by magnetic beads based ECL platform to confirm the presence of targets. Through this approach, as low as 10 aM synthetic hly gene targets and about 0.0002 ng/μl of genomic DNA from L. monocytogenes can be detected, the ability to detect at such ultratrace levels could be attributed to the powerful amplification of HRCA and the high sensitivity of current magnetic bead based ECL detection platform.     

Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes

Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucial components of the epidemic. To detect and discriminate these two closely related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Two padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs) located at the inter-generic spacer 2 region and large subunit of the rRNA respectively, which allows discrimination between the two species. Thirty-four strains of G. clavigera and twenty-five strains of L. longiclavatum representing a broad geographic origin were tested with this assay. The HRCA results were largely in agreement with the conventional identification based on morphology or DNA-based methods. Both probes can also efficiently distinguish the two MPB-associated fungi from other fungi in the MPB, as well as other related fungi in the order Ophiostomatales. We also tested this diagnostic method for the direct detection of these fungi from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is a rapid, sensitive and reproducible method for the identification and detection of these ophiostomatoid fungi.     

Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification

A simple isothermal nucleic-acid amplification reaction, primer generation–rolling circle amplification (PG–RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60°C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, ‘primers’ are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG–RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of ‘primers’ are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (~60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays.     

Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR

AIMS: The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains. METHODS AND RESULTS: A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain. One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids. The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR. CONCLUSIONS: The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis. The technique is therefore a rapid and cost-effective detection method. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen. The method can be used as a model system for the detection of other Agrobacterium pathogens.     

Detection of denitrification genes by in situ rolling circle amplification‐fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells

A target‐primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single‐copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene to target both DNA strands; the target DNA was cut by a restriction endonuclease close to the probe binding sites, which subsequently were made accessible by 5′‐3′ exonucleolysis. After hybridization, the padlock probe was circularized by ligation and served as template for in situ RCA, primed by the probe target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal‐to‐noise ratio but low detection frequency (up to 15% for single‐copy genes and up to 43% for the multi‐copy 16S rRNA gene). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA‐FISH was demonstrated on activated sludge by the differential detection of two types of nirS‐defined denitrifiers; one of them was identified as Candidatus Accumulibacter phosphatis by combining in situ RCA‐FISH with 16S rRNA‐targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA‐FISH will allow to link metabolic potential with 16S rRNA (gene)‐based identification of single microbial cells.     

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.