实时荧光定量PCR技术及其在植物转基因产品检测中的应用

实时荧光定量PCR技术因其实时、快速、高效和准确定量的优点,已经广泛用于转基因产品定量检测。本文介绍荧光定量探针技术的原理、特点及其在植物转基因产品检测中的应用情况。     

转基因产品免疫学检测技术研究进展

目前转基因产品的检测主要有基于DNA的检测方法以及基于蛋白质的免疫学检测方法,简述了免疫学检测方法中的免疫印迹法(Western blotting)、酶联免疫吸附试验(ELISA)和试纸条法的原理、特点、应用例子以及存在问题。对一些新的方法如免疫PCR法、生物传感器法也略作介绍。     

转基因植物核酸成分检测技术研究进展

首先对转基因核酸成分检测的靶序列特征进行了阐述,对转基因植物核酸成分的定性、定量检测技术研究进展进行了综述,包括基于PCR的检测技术、基于等温核酸扩增的检测技术、基因芯片检测技术、基于高通量测序和新型转基因核酸检测技术(如生物传感器技术、毛细管电泳技术和纳米刻度技术等),重点介绍了各种检测技术的原理、特点、研究现状和发展动态,并对各种方法的优缺点进行了比较。     

免疫层析试纸条技术及其在转基因检测中的应用

转基因检测是转基因生物安全监管的重要环节。目前需要一种适合于大规模筛查的快速检测转基因成分的方法,以扩大转基因生物安全的监测范围。免疫层析试纸条是一种快速免疫分析技术,是目前常用的快速检测方法之一,具有操作简单、样品量少、检测快速、成本低等优点,已在医学、食品和环境等多个领域广泛应用。介绍了免疫层析试纸条的组成结构、检测原理及免疫标记物,并总结了其在转基因检测方面的研究进展。     

遗传修饰工程体的生态安全性

遗传修饰工程体的生态安全性引起了人们的广泛关注。本文对转基因植物外源基因逃逸、对非靶标生物的影响、抗生素抗性基因的安全性以及生防工程菌的生态安全性等问题作了讨论。     

出入境转基因产品及其分子检测现状与展望

随着转基因产品在全球的迅速推广,包括我国在内的很多国家都建立了转基因标识制度。各检验检疫口岸应转基因产品生产企业、食品制造商、消费者等多方面需要,相继开展了转基因产品的检测工作。准确可靠的转基因产品检测技术是各国检疫检疫单位的共同需求。转基因产品的检测主要有两大类方法,一类是DNA水平上的检测,另一类是蛋白质水平上的检测。多个发达国家也相继成立专门机构或部门,负责转基因产品生物检测技术标准化工作。国际上对转基因产品的检测工作有向委托鉴定方向发展的趋势。我们简要综述了出入境转基因产品及其分子检测现状。     

实时荧光定量PCR方法快速检测转基因大豆

针对转基因大豆中普遍含有的35S启动子进行引物设计,以双链DNA染料SYBR GreenⅠ为荧光标记物,利用实时荧光定量PCR方法对大豆样品进行检测。该法检测转基因大豆的检测低限为0.005 nmol/L的35S启动子,线性范围达3个数量级,可快速区分转基因大豆和非转基因大豆,具有快速、简便、灵敏、安全、高通量、低成本等优点,可推广用于转基因植物产品的快速定量检测。     

转基因植物快速检测方法的研究

本试验对转基因植物检测中的DNA提取和PCR扩增程序作了改进。经试验,本研究建立的DNA快速提取法与目前广泛使用的CTAB法相比更为简便,快速和经济,提取的DNA质量主扩增效果无明显差异,可用于多种转基因植物,多种植物组织的DNA提取,利用复合PCR法可在同一反应管中同步检测35N,NOS及CP4－EPSPS基因,明显提高了检测效率。应用本试验建立的DNA快速提取－复合PCR扩增－银染检测技术可在6小时内得出结果,达到了快速,简便,灵敏,可靠的检测目的。     

绿色荧光蛋白及其在GMOs 生态监测中的应用

绿色荧光蛋白 (green fluorescent protein, GFP)是在海洋无脊椎动物水母(Aequorea victoria)中获得的一种由238个氨基酸组成的多肽。该多肽通过翻译后加工形成生色基团, 产生稳定的荧光, 而且这种荧光很容易被检测。GFP作为动、植物以及微生物基因工程研究上的一种广泛的选择标记, 具有检测灵敏度高、操作简便、不需要添加任何底物或辅助因子等优点, 更重要的是利用GFP可对GMOs进行快速、原位、实时、活体监测。本文概括介绍了GFP的特性、改造及其检测, 并从生态学角度论述了GFP在GMOs生态监测研究中的应用及其发展前景。     

绿色荧光蛋白及其在GMOs生态监测中的应用

绿色荧光蛋白（green fluorescent protein,GFP）是在海洋无脊椎动物水母（Aequorea victoria）中获得的一种由238个氨基酸组成的多肽。该多肽通过翻译后加工形成生色基团,产生稳定的荧光,而且这种荧光很容易被检测。GFP作为动、植物以及微生物基因工程研究上的一种广泛的选择标记,具有检测灵敏度高、操作简便、不需要添加任何底物或辅助因子等优点,更重要的是利用GFP可对GMOs进行快速、原位、实时、活体监测。本文概括介绍了GFP的特性、改造及其检测,并从生态学角度论述了GFP在GMOs生态监测研究中的应用及其发展前景。     

Diffusion, Transport, and Cell Membrane Organization Investigated by Imaging Fluorescence Cross-Correlation Spectroscopy

Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid “platforms”). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), a widely used spectroscopic technique to measure membrane dynamics, has the required time resolution but lacks imaging capabilities. A promising solution is the recently introduced method known as imaging total internal reflection (ITIR)-FCS, which can probe diffusion phenomena in lipid membranes with good temporal and spatial resolution. In this work, we extend ITIR-FCS to perform ITIR fluorescence cross-correlation spectroscopy (ITIR-FCCS) between pixel areas of arbitrary shape and derive a generalized expression that is applicable to active transport and diffusion. ITIR-FCCS is applied to model systems exhibiting diffusion, active transport, or a combination of the two. To demonstrate its applicability to live cells, we observe the diffusion of a marker, the sphingolipid-binding domain (SBD) derived from the amyloid peptide Aβ, on live neuroblastoma cells. We investigate the organization and dynamics of SBD-bound lipid microdomains under the conditions of cholesterol removal and cytoskeleton disruption.     

Determination of Dissociation Constants in Living Zebrafish Embryos with Single Wavelength Fluorescence Cross-Correlation Spectroscopy

The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (K_D) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42^G12V which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42^T17N. While Cdc42^G12V binds to IQGAP1 with an apparent K_D of ∼100 nM, Cdc42^T17N has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and K_D from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions.     

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。     

利用MPCR方法快速检测植物转基因背景

利用复合ＰＣＲ（Ｍｕｌｙｉｐｌｅｘ ＰＣＲ－ＭＰＣＲ）技术对植物的转基因背景进行检测。经过对ＤＮＡ方法的选择,对各种ＰＣＲ程序的比较以及对引物的修饰,建立了一种快速检测植物转基因情况的技术;利用该技术对５个大豆样品,６个豆粕样品进实验检测,同时利用普通ＰＣＲ方法对上述样品进行检测,两者的结果完全符合。     

Metabolism of Zearalenone by Genetically Modified Organisms Expressing the Detoxification Gene from Clonostachys rosea

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101. An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45°C and pH 10.5 (k_cat at 30°C, 0.51 s⁻¹). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the k_cat was highest with zearalanone (1.5 s⁻¹) and lowest with β-zearalenol (0.075 s⁻¹). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN. While recombinant E. coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, β-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykkuäne, Appl. Environ. Microbiol. 68:3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.     

基于CRISPR/Cas原理的转基因产品检测技术研究进展

随着转基因产品商业化种植面积不断增加、国际贸易日趋频繁,对转基因生物安全管理提出了更高的要求。转基因产品检测技术作为安全评价的关键环节,逐渐引起了各国政府的关注。目前,针对转基因产品的快速检测方法层出不穷,但这些检测方法对于设备、试剂和专业的实验人员均有较高的要求。因此,为了有效支撑转基因相关产业的发展和管理,亟需建立一种高灵敏度、高特异性及高效的转基因检测技术。基因组编辑技术是近年来迅速发展的一类遗传修饰技术,其代表技术——CRISPR/Cas技术,更是极大地推动了生物技术的发展。CRISPR/Cas技术除了被应用于基因编辑领域,也逐渐被应用于核酸分子检测领域。基于此,以转基因产品检测技术为立足点,从CRISPR/Cas的检测原理、检测效果等技术层面分析了CRISPR/Cas检测技术发展的必然性,并对其在转基因产品检测上的应用前景进行展望,旨在为我国转基因产品快速检测和有效监管工作提供资料,对于保障我国转基因产品贸易的顺利进行具有重要意义。     

电化学发光PCR技术检测转基因植物

随着转基因植物种类的增多,转基因植物的检测也成了当今的热门话题.电化学发光法是将电化学与化学发光两种高灵敏度方法相结合,实现了检测的高效、准确、无毒害.电化学发光PCR法首次将电化学发光技术、PCR技术和双探针杂交技术结合起来,用于检测CaMV(cauliflower mosaic virus)35 S启动子,从而判断其是否含有转基因成分.PCR产物与生物素标记的探针杂交,可以起到筛选的作用;与三联吡啶钌标记的探针杂交则可用于电化学发光检测.两种探针同时与转基因样品PCR产物杂交,使结果避免假阳性的影响而更加准确.实验表明：此方法可以准确地检测到35 S启动子的存在.该方法灵敏度高,可靠性强,操作简便,结果准确,有望成为一种高效的转基因检测方法.     

Detection of a Single Genetically Modified Bacterial Cell in Soil by Using Charge Coupled Device-Enhanced Microscopy

Genes encoding bioluminescence from Vibrio harveyi were cloned into Pseudomonas syringae pv. phaseoli-cola, resulting in high levels of bioluminescence. After inoculation of sterile and nonsterile soil slurries with bioluminescent P. syringae, cells could not be identified by conventional light microscopy. However, when we used charge coupled device-enhanced microscopy, bioluminescent single cells were detected easily in dark fields despite masking by soil particulate matter, and in addition, the extent of competition from indigenous soil bacteria could be monitored. The technique which we describe offers great potential for tracking and determining the spatial distribution of genetically marked microorganisms in the environment.