分子诊断实验室去除核酸污染的方法学研究

核酸检测因具有良好的灵敏度和特异性而被广泛应用于体外诊断、动植物商品检疫、法医鉴定等领域。然而操作过程中易受到核酸污染导致的假阳性结果,严重影响了检测准确性。因此寻找一种有效的防止和清除核酸污染的方案对于实验室正常运转及保障检测结果的可靠性具有重要意义。文中比较了几种不同清除核酸污染的方法,确认了84消毒液和PCRguard试剂可有效清除液体中及不同材质表面的核酸污染。另外,84消毒液和PCRguard试剂配合使用,可很好地解决核酸气溶胶污染。研究结果对于分子诊断实验室日常预防核酸污染及清除已发生的气溶胶污染提出了解决方案。     

多重核酸检测技术研究进展

核酸检测技术因其快速、灵敏、特异、准确等优点,被广泛应用于细菌、真菌、病毒、寄生虫的快速检测和鉴定,以及疾病的早期筛查与诊断中。随着生物检测技术的发展,基于核酸的多重检测技术在核酸诊断领域发挥了越来越重要的作用,主要包括以多重PCR、核酸等温扩增、基因芯片为基础的多重核酸检测技术,这些技术可对多个靶标进行同时检测,具有快速、高通量、样品消耗少等特点。本文扼要介绍这些技术的原理,及其在病原检测、疾病诊断等方面的应用。     

悬浮芯片在核酸和蛋白质检测中的应用

悬浮芯片是近年来兴起的一种新型检测技术,不同于固相基因芯片,它整合了高分子化学、分子生物学、免疫学、激光检测、微流体、高速数字信号处理、计算机分析等方面的先进技术,能够对少量样本进行高通量的定性、定量检测。主要综述了悬浮芯片技术的基本原理,并概要介绍了其在核酸和蛋白质检测中的应用。悬浮芯片技术在核酸和蛋白质检测中有着显著的优点,如高通量、操作简便、重复性好、灵敏度高、线性范围宽等,不但可以广泛应用于科学研究领域,而且还将逐渐普及于临床诊断实验室,具有广阔的应用前景。     

简化核酸提取过程在病原体核酸检测中的应用现状和发展趋势

简化核酸提取过程相较于经典的试剂盒法和全自动核酸提取技术,凭借其简便的操作步骤、便携易用的仪器设备和简单的人员培训等优势在病原体核酸检测中得到广泛应用,为实现病原体核酸的快速检测和现场检测(Point-of-care testing,POCT)奠定了良好的基础。本文比较了不同的核酸提取方法,对简化核酸提取过程的方法学进行综述,讨论其在病原体核酸检测领域中的应用现状,并展望其发展趋势。     

基于CRISPR的快速灵敏便捷分子检测

快速灵敏检测技术对疾病防控必不可少。特别是新冠疫情暴发以来,人们深刻认识到快速灵敏检测技术的重要性。近年来,以CRISPR/Cas为代表的基因编辑技术带来了生物技术革命性的进步。CRISPR的核酸检测技术因其快速、准确、灵敏、经济等特点,正在引发分子诊断革新,并已被成功应用于传染病、遗传病、肿瘤基因突变诊断,以及食品安全等领域。本文归纳了基于CRISPR的多种核酸检测体系及应用,并对未来CRISPR核酸检测发展趋势及结合人工智能的智能化检测进行了展望。     

核酸分子的化学发光标记和化学发光检测

非同位素标记是核酸分子杂交和检测的关键技术,在已知的各种非同位素标记检测方法中,化学发光标记和检测以其灵敏度高,安全无害和操作简便等优点,愈来愈受到重视,本对核酸分子的化学发光标记和核酸杂交分子的化学发光检测作一简要介绍。     

生物条形码检测技术及其研究进展

生物条形码检测技术是利用磁场作用收集"金纳米颗粒-被检物-磁性微球"复合物,再释放其中的条形码链,然后可选用多种方法对条形码链进行下一步检测。生物条形码检测技术在蛋白质及核酸的检测方面显示出极高的灵敏度,在不依赖酶反应的情况下,检测蛋白质可以达到10-18mol水平,同时在核酸检测方面也能达到PCR反应的灵敏度。该技术是一种新型的诊断技术,在临床诊断方面有着广泛的应用前景。     

生物素标记核酸探针的分子杂交技术及其应用

核酸分子杂交是基于核酸链间互补碱基对的特异性结合,测定核酸碱基顺序同源性的一种现代技术,是基因工程、分子遗传及医学诊断中常用的方法。该法较常规诊断法灵敏度高、特异性强,目前,已广泛应用于重组DNA的筛选、基因分祈、染色体基因定位及病毒病诊断等方面。     

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。     

用于新型冠状病毒检测的核酸等温扩增技术研究进展

核酸检测作为新型冠状病毒肺炎(COVID-19)筛查诊断和病情监测的主要手段,在疫情防控中发挥了重要作用。虽然实时荧光定量PCR被认为是新型冠状病毒(SARS-CoV-2)核酸检测的金标准,但其依赖荧光定量PCR仪且扩增检测时间较长,难以实现现场快速检测。因此许多基于核酸等温扩增的SARS-CoV-2检测方法相继诞生。等温扩增对仪器温控要求不高,通过与微流控芯片和可视化检测技术结合,可进一步简化操作、降低成本,为SARS-CoV-2现场快速筛查提供有力的技术支撑。本文围绕已报道的SARS-CoV-2等温扩增检测方法原理、检测性能及优缺点进行探讨,为进一步发展SARS-CoV-2现场快速检测平台提供参考。     

Development Of A Nucleic Acid Lateral Flow Strip For Detection Of Hepatitis C Virus (Hcv) Core Antigen

The object of this study was to develop a simple, rapid, specific, and highly sensitive method to detect HCV core antigen. A nucleic acid aptamer was designed with the high specificity and sensitivity in a nucleic acid lateral flow strip to compete with HCV core antigen and DNA probes. The lower detection limit of the test strip was calculated to be 10 pg/mL with the scanner and 100 pg/mL with naked eyes. Results showed that there were no cross-interactions with other proteins such as HCV NS3, E1/E2 antigens, HIV p24 antigens, or BSA proteins (HCV unrelated protein). When the viral load exceeded 10⁴ copies/mL, the positive coincidence rates of ELISA and strip detection, when compared with the HCV RNA assay, were 98.44% and 97.28%, respectively. The results indicated that the ELISA detection and strip assay were in good agreement with the measured value. The results indicated that a nucleic acid lateral flow strip was a simple, rapid, specific, highly sensitive, and cost-effective field-based method for detecting HCV core antigen. The strip assay is an acceptable alternative to diagnose HCV core antigen and to investigate its epidemiology in clinical laboratories lacking specialized equipment and skills.     

核酸检测技术在水产养殖病原诊断中的应用

目前核酸检测技术在水产养殖疾病诊断中已广泛应用。现就国内外研究发展动向,对分子杂交技术、PCR技术、PCR分子杂交联用技术、PCR免疫学联用技术、16SrRNA技术和限制性酶切技术等方法进行综述,详细阐明了各个技术的原理、特点及应用。病原体遗传背景的研究状况为决定是否应用这些方法的关键。     

食源性病毒核酸恒温检测技术研究进展

食源性病毒已成为全球引发食品安全事件的重要病原,对新型检测技术的不断发展提出了严峻的挑战。早期PCR技术在病原检测领域中的应用,推动了对食源性病毒的全面认识。近年来核酸恒温检测技术发展迅速,包括环介导等温扩增技术、重组酶聚合酶扩增技术、核酸序列依赖性扩增技术、链置换扩增技术、滚环扩增技术等,在抗复杂基质干扰、装备要求低以及可现场实时检测等方面具有明显的技术优势,已成为食源性病毒检测领域的热点研究方向。因此,本文对近年来食源性病毒核酸恒温检测技术的原理、应用、优缺点等方面进行综述,并对未来发展方向进行了展望。     

Nucleic acid probes for the food industry

Recombinant DNA technology has revolutionised microbiological analysis by providing rapid and sensitive methods for the direct detection and identification of organisms. These advances have been most successfully applied to basic research and to clinical microbiology. Continued improvements in the techniques and in assay formats have now brought them to the stage where they could be useful on an industrial scale. The use of nucleic acid techniques for the identification of bacteria of importance to the food industry is discussed with emphasis on developments in practical applications.     

Nucleic acid structure and sequence probing using fluorescent base analogue tC(O)

The fluorescent cytosine analog tC(O) is on average the brightest probe of its kind and, moreover, it introduces minimal perturbations to the normal secondary structure of DNA. Here several ways of how tC(O), with an advantage, can be used as a local fluorescent probe in nucleic acid systems are presented. Most importantly, we show that tC(O) is an excellent probe for the detection of individual melting processes of complex nucleic acid structures containing a large number of separate secondary structure motifs. Since conventional UV-melting investigations merely monitor the global melting process of the whole nucleic acid structure, e.g. multi-hairpin systems in RNA/DNA, and thus is incapable of estimating individual melting transitions of such systems, tC(O) represents a new method of characterization. Furthermore, we find that tC(O) may be used to detect bulges and loops in nucleic acids as well as to distinguish a matched base-pair from several of the mismatched.     

Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay

Up-converting Phosphor Technology (UPT) particles were used as reporters in lateral-flow (LF) assays to detect single-stranded nucleic acids. The 400-nm phosphor particles exhibit strong visible luminescence upon excitation with infrared (IR) light resulting in the total absence of background autofluorescence from other biological compounds. A sandwich-type hybridization assay was applied using two sequence-specific oligonucleotides. One of the oligonucleotides probes was covalently bound to the UPT particle (reporter) for direct labeling and detection, whereas the second oligonucleotide probe contained biotin for capture by avidin during LF. The whole procedure of hybridization, UPT-LF detection, and analysis required a minimum time of 20 min. Moreover, aiming at minimal equipment demands, the hybridization conditions were chosen such that the entire assay could be performed at ambient temperature. During lateral flow, only targets hybridized to both capture and detection oligonucleotide were trapped and detected at an avidin capture line on the LF strip. Analysis (IR scanning) of the strips was performed in an adapted microtiter plate reader provided with a 980-nm IR laser for excitation of the phosphor particles (a portable reader was also available). Visible luminescence was measured and presented as relative fluorescence units (RFU) allowing convenient quantitation of the phosphor signal. With the assay described here as little as 0.1 fmol of a specific single-stranded nucleic acid target was detected in a background of 10 microg fish sperm DNA.     

Postcolumn nucleic acid intercalation for the fluorescent detection of nucleic acids using ion pair reverse phase high-performance liquid chromatography

Here we report a simple and effective procedure enabling the fluorescent detection of nucleic acids following the rapid, high-resolution separation using ion pair reverse phase chromatography. This approach uses postcolumn nucleic acid intercalation of fluorescent dyes with subsequent fluorescent detection, demonstrating more than a 1000-fold increase in sensitivity in the detection of nucleic acids when compared with traditional UV detection. Moreover, a wide range of intercalating dyes can be incorporated, including those known to disrupt the structure of the nucleic acids, thereby enabling the sensitive detection of DNA and RNA with no adverse effect on resolution of the nucleic acids during ion pair reverse phase chromatography. In addition, such approaches allow one to readily distinguish single-stranded DNA from double-stranded DNA following their separation using ion pair reverse phase high-performance liquid chromatography.     

利用纳米粒子的光学性质高敏感性检测核酸的研究进展

将纳米粒子的光学性质应用于核酸检测是纳米技术应用的一个重要内容,其检测敏感性可达到10-15mol/L(fmol/L,飞摩尔浓度)或亚飞摩尔浓度水平,而且检测成本低、时间短、具有和PCR类似的敏感性、稳定性好,不需要复杂、大型、昂贵的仪器,使得核酸检测可以走出专业的实验室,在野外、家庭中进行。本文主要综述纳米粒子和核酸的偶联,以及利用纳米粒子的光吸收性质、光散射性质、高强度荧光性质进行核酸检测的应用进展。     

高灵敏可视化核酸试纸条法快速检测HBV DNA

目的：本研究旨在建立一种基于试纸条的快速、灵敏及可视化检测乙型肝炎病毒核酸的方法。方法：利用聚合酶链反应扩增乙肝病毒的保守区,其中上、下游引物的5＇端分别修饰异硫氰酸荧光素和生物素。核酸试纸条上的胶体金以及检测线处分别标记有链霉亲和素以及抗荧光素抗体。将扩增产物与展开液混合后点样,10 min后即可用肉眼判读结果。在优化了展开液成分、上样体积以及上样浓度之后,对该方法的灵敏度进行了评价。最后收集15例阴性样本及33例HBsAg阳性样本,按血清标志物结果进行分类后使用核酸试纸条进行检测,并与实时荧光PCR的结果进行了比较。结果：试纸条检测乙肝病毒核酸的灵敏度为250copies/mL。在临床样本的测定中,该方法与实时荧光定量PCR的特异性均为100%。且两种方法检测不同血清标志物类型的阳性检出率无差异。结论：核酸试纸条技术能够用于乙肝病毒核酸的可视化检测,与实时荧光PCR相比检测速度快,具有较好的灵敏度和特异性,适合流行病学调查以及在基层医院体检使用。