用重组酶介导扩增技术快速扩增核酸

体外核酸快速扩增技术是一种可使微量核酸在体外高效快速扩增的技术, 自问世以来, 被广泛应用于分子生物学、医学和法理鉴定等领域, 并被不断改进, 以使其功能和适应性更为广泛. 重组酶介导扩增法是在现有体外核酸扩增原理的基础上发展起来的恒温体外快速扩增核酸技术. RAA法利用重组酶、单链结合蛋白和DNA聚合酶代替了传统PCR的热循环解链过程, 实现了在37℃恒温下的核酸快速扩增, 无需特殊的辅助仪器, 对操作人员的要求也不高, 具备简单、节能、便携、快速等特点, 有望在不远的将来取代传统的热循环PCR反应.     

同步PCR技术及其在植物核酸分子定量中的应用

同步PCR是一种集生化、光电和计算机技术于一体的封闭式DNA扩增系统,采用荧光染料将扩增与检测过程结合在一起,实现了在PCR过程中在线显示PCR反应,通过检测荧光强度来绝对定量起始模板的拷贝数.该技术大大简化和加速了核酸分子的定量过程,不仅快速、灵敏、准确、重复性好,而且很容易计算出待测样品中核酸分子的绝对起始拷贝数.同微阵列等分子生物技术一起,同步PCR技术将会在功能基因解析和病害分子诊断等方面发挥重要作用.本综述除了介绍同步PCR技术的原理和应用外,还介绍了定量拟南芥Aux/IAA基因的转录水平的实验,并就同步PCR操作过程中的问题进行了讨论.     

同步PCR技术及其在植物核酸分子定量中的应用

同步PCR是一种集生化、光电和计算机技术于一体的封闭式DNA扩增系统,采用荧光染料将扩增与检测过程结合在一起,实现了在PCR过程中在线显示PCR反应,通过检测荧光强度来绝对定量起始模板的拷贝数。该技术大大简化和加速了核酸分子的定量过程,不仅快速、灵敏、准确、重复性好,而且很容易计算出待测样品中核酸分子的绝对起始拷贝数。同微阵列等分子生物技术一起,同步PcR技术将会在功能基因解析和病害分子诊断等方面发挥重要作用。本综述除了介绍同步．PCR技术的原理和应用外,还介绍了定量拟南芥,Aux／正4,4基因的转录水平的实验,并就同步PCR操作过程中的问题进行了讨论。     

科技信息

荧光定量PCR技术是一种先进的定量核酸检测分子诊断技术,在西方国家广泛用于乙肝HBV,丙肝HCV,艾滋病HIV,性病STD等传染病及肿瘤性疾病快速早期诊断,并作为一种先进手段用于多种生物学及医学研究,在我国现行禽流感诊断国家标准中也规定了要用该方法检测核酸确诊。荧光定量基因扩增PCR检测系统是实现荧光定量PCR技术的核心设备,目前国内主要依赖进口,价格大约是普通PCR仪的十倍。西安天隆科技有限公司通过与西安交通大学教育部生物医学信息工程重点实验室生物医学及分子光子学中心产学研结合,自主研发的荧光定量基因扩增PCR检测系…     

体外核酸快速扩增技术的发展和不断创新

利用聚合酶链式反应(PCR)进行的核酸体外扩增是1983年开始发展起来的一项革命性技术,目前已被广泛运用于现代化的农业和医学以及食品工业等领域,特别是在人类认知基因和基因组的过程中,体外核酸扩增技术做出了卓越的贡献。最初,体外核酸扩增技术主要是利用耐高温的DNA聚合酶(Taq酶),这样就使核酸的体外扩增反应可以在热循环中进行。但因需要使用昂贵的设备和消耗大量的电力,其成本和应用范围都受到一定的限制。之后,恒温体外核酸扩增悄然兴起,这改变了传统扩增技术的局限性,使核酸的体外扩增更加简单和方便。重组酶介导扩增(RAA)法是一种最新型的恒温体外核酸扩增技术,该系统的显著优点在于它在常温下就能实现DNA解链并快速扩增(15~30min完成),反应快速、专一性好、灵敏度高,还可用于定时定量的结果分析。     

植物病原真菌分子检测技术的研究进展

真菌是一类非常重要的植物病原菌。对这类病原菌进行分子快速检测是病害早期诊断、检疫、监测、预测预报和病害防治等工作的重要基础。本综述详细介绍与评述了一些常见的以PCR技术为基础的检测技术以及近年来发展起来的一些新技术如环介导等温扩增和核酸滚环扩增技术在植物病原真菌分子检测中的应用,旨在对该研究领域的进展有一个全面的了解。     

基于微流控芯片的核酸等温扩增技术研究进展

核酸等温扩增技术是一种在恒温体系内对核酸进行高效扩增的分子扩增技术,它能够在短时间内实现目的基因的指数增长。微流控芯片(microfluidic chip)技术是把研究样品制备、核酸富集、纯化和检测等多个操作步骤集成到一块"微型化"的芯片上,经自动化处理,得出实验结果,即"样品进,结果出"。将核酸等温扩增技术与微流控芯片相结合,不仅可以实现核酸快速扩增,还可以降低对实验器材的依赖。在床边即时诊断、病原体快速筛查中具有广阔的应用前景。综合国内外相关研究报道,综述了各种等温扩增技术原理,以及基于微流控芯片的核酸等温扩增技术应用,展望了集成化微流控芯片的发展趋势和应用前景。     

基于微流控芯片的等温扩增技术北大核心CSCD

基于聚合酶链式反应(polymerase chain reaction,PCR)的核酸扩增技术是分子诊断领域的金标准,然而PCR往往包含多个反应温度,涉及长时间的循环升降温过程,且需要在复杂热循环仪中完成,这些都限制了其在现场即时检测(point-of-care testing,POCT)中的应用。与传统PCR相比,等温扩增依靠恒定反应温度,反应时间短,检测装置简单,能够提供更加方便、快捷的核酸检测。基于微流控技术的等温扩增检测,通过兼顾微流控与等温扩增两者的优势,能够为POCT分子诊断提供更具竞争力的平台。例如,在新型冠状病毒肺炎(COVID-19)疫情防控中,多种形式的POCT等温扩增检测展示了其独特优势。文中首先归纳总结了典型的等温扩增技术及其检测方法,然后对不同类型的等温扩增微流控系统进行了分类总结与分析(如功能定位、结构组成、流体控制、系统特点等),最后总结了等温扩增微流控系统在新冠病毒(SARS-CoV-2)等不同病原体检测领域中的应用,并对等温扩增与CRISPR基因编辑等其他新型技术的相互结合进行了介绍与展望。     

PCR增强剂在核酸体外扩增检测技术的研究进展

在各种高致病性病原体、禽流感病毒、食源性微生物等引起的疾病随时大规模流行的背景下,利用聚合酶链式反应（polymerase chain reaction,PCR）技术对第一例或第一波病例的快速实验室诊断显得尤为重要,同时发展出多种以PCR技术为基础的检测技术以便更加快速、高通量、敏感地对疾病进行诊断、预防或预测。然而,在实际病原体检测中,常常出现灵敏度低、准确性差的结果。PCR增强剂是在PCR及PCR衍生技术中添加的一类物质,其可从产率、特异性、灵敏度等方面提高核酸扩增性能,从而优化核酸检测,解决病原体检测的应用瓶颈,为第一例病原体检出节约宝贵的时间。结合以PCR为基础的核酸体外扩增检测技术对PCR增强剂在其中的应用、优缺点、作用机理进行介绍,以期为病原体核酸检测的实际应用提供一些参考。     

任意引物PCR及其应用研究进展

任意引物PCR技术又称为随机扩增多态性DNA技术,它是在PCR技术基础上发展起来的一项分子检测技术。它具有简便、快速,一套引物可用于多个物种的分析,不需预知分析对象的核酸序列,可以显示差异表达基因等特点,已广泛应用于病原微生物的分型鉴定、物种亲源关系分析、遗传育种研究和特异表达基因的克隆与鉴定等方面。     

Identification of a single genome by electron paramagnetic resonance (EPR) with nitroxide-labeled oligonucleotide probes

Nitroxide-labeled nucleic acids are used as a molecular size sensor to identify as few as one genome under polymerase chain reaction (PCR) conditions by electron paramagnetic resonance (EPR) spectroscopy. DNA identification is based on differences in the EPR spectra of mono-nitroxide-labeled nucleic acids. The experimental data imply that rapid DNA identification can be achieved in many systems by EPR at the molecular level.     

A powerful but simple technique to prepare polysaccharide-free DNA quickly and without phenol extraction

A protocol for the rapid isolation of polysaccharide-free DNA without phenol extraction is described. Total nucleic acids are first extracted according to Guillemaut and Maréchal-Drouard (1992) and then bound to an ion exchanger while contaminants not bound are washed away. The entire procedure can be carried out in Eppendorf tubes. The highly purified DNA obtained is suitable for restriction by the common endonucleases and serves as a substrate for PCR.     

Pre-PCR processing

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.     

Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids

Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02–1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5′-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.     

硅对PCR过程的抑制作用研究

探讨不同氧化程度的硅材料对PCR扩增的抑制作用及其机理。将不同氧化程度的硅纳米颗粒加入PCR反应液中,使其与Taq酶、模板等充分接触,通过离心将硅纳米颗粒沉降在管壁上,取出上清或保留硅纳米颗粒上机扩增,扩增产物采用凝胶电泳法检测。结果表明,随着硅材料表面面积与PCR反应液体积之比的增大,核酸扩增效率将明显下降,并且在所研究的范围内,氧化程度高的硅材料对PCR过程抑制作用更强;通过对抑制作用机理进行初步的实验研究,表明硅材料对PCR反应液中的Taq酶的吸附是导致抑制现象产生的主要原因,而对模板的吸附影响较小;并且,反应管内是否保留硅材料对核酸扩增影响较小,硅材料没有明显的直接化学抑制作用。     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

基因扩增产物的固相杂交-酶联显色方法的建立

建立基于基因扩增技术的简便、快速的病毒核酸定量检测方法．将标记有生物素的寡核苷酸引物所扩增的病毒基因产物,与通过共价键结合在微孔反应板上的特异性探针进行快速杂交,然后通过辣根过氧化物酶标记的抗生物素进行酶联显色,读取光密度值．应用本方法对血清中乙型、丙型肝炎病毒核酸定量检测,灵敏度分别可达１－５拷贝／反应．此方法简便、快速、特异性好、敏感性高、半定量指标客观,可广泛应用于肝炎病毒感染的临床诊断和疗效评价．     

Research progress in the detection of common foodborne hazardous substances based on functional nucleic acids biosensors

With the further improvement of food safety requirements, the development of fast, highly sensitive, and portable methods for the determination of foodborne hazardous substances has become a new trend in the food industry. In recent years, biosensors and platforms based on functional nucleic acids, along with a range of signal amplification devices and methods, have been established to enable rapid and sensitive determination of specific substances in samples, opening up a new avenue of analysis and detection. In this paper, functional nucleic acid types including aptamers, deoxyribozymes, and G-quadruplexes which are commonly used in the detection of food source pollutants are introduced. Signal amplification elements include quantum dots, noble metal nanoparticles, magnetic nanoparticles, DNA walkers, and DNA logic gates. Signal amplification technologies including nucleic acid isothermal amplification, hybridization chain reaction, catalytic hairpin assembly, biological barcodes, and microfluidic system are combined with functional nucleic acids sensors and applied to the detection of many foodborne hazardous substances, such as foodborne pathogens, mycotoxins, residual antibiotics, residual pesticides, industrial pollutants, heavy metals, and allergens. Finally, the potential opportunities and broad prospects of functional nucleic acids biosensors in the field of food analysis are discussed.     

Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference

Application of polymerase chain reaction (PCR) techniques has developed significantly from a qualitative technology to include powerful quantitative technologies, including real-time PCR, which are regularly used for detection and quantification of nucleic acids in many settings, including community analysis where culture-based techniques are not suitable. Many applications of real-time PCR involve absolute quantification which is susceptible to inaccuracies caused by losses during DNA extraction or inhibition caused by co-extracted compounds. We present here an improvement to this approach involving the addition of an artificial internal standard, prior to nucleic acid extraction. The standard was generated by in-situ mutagenesis from an E. coli template to ensure it both did not amplify with bacterial primers used for quantification and was short enough to minimise possible interference with other analyses. By estimating gene target copies by relative abundance, this approach accounts for both loss during extraction and inhibition effects. We present a novel application of relative real time PCR, using the internal standard as a reference, allowing accurate estimation of total bacterial populations both within and across a wide range of soils and demonstrate its improvement over absolute quantification by comparison of both approaches to ester linked fatty acid analysis of the same soils.