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。     

转基因产品检测方法概述

随着转基因技术的快速发展,转基因生物及其产品日益增多,但其安全性问题引起了国际社会的广泛关注。转基因产品的检测已纳入国内外检验检疫部门的检测项目,采用的检测方法是建立在已商品化生产的转基因生物外源基因的构建及表达情况的基础上的,包括蛋白质检测和DNA检测方法。蛋白质检测方法有ELISA、试纸条、免疫PCR等,DNA检测方法有PCR、多重PCR、PCR-EUSA、PCR-GeneScan、荧光定量PCR、基因芯片等。     

香茄环斑病毒HC—RT—PCR—ELISA检测

番茄环斑病毒（ToRSV)是我国对外检疫一类有害生物。目前国内尚无存在的报道。PCR技术是一种快速灵敏的植物病毒检测方法,但核酸内的聚合酶抑制物会导致漏检现象。而只通过凝胶电泳进行结果判定会出现假阳性,这两方面限制了PCR技术在对外检疫中的应用,利用共价结合在PCR管壁上的引物特异性杂交诱捕核酸粗提液中的靶标核酸,洗掉杂质及抑制物质,在同一管内作RT-PCR,凝胶电泳检测液相产物的同时对固相产物进行杂交检测。提高了结果的可靠性及灵敏度。利用所建立的HC-RT-PCR-ELISA成功地从法国进口葡萄苗中检出ToRSV。本方法可用于其他植物病毒及转基因产品的检测。     

番茄环斑病毒HC-RT-PCR-ELISA检测

番茄环斑病毒（ToRSV）是我国对外检疫一类有害生物,目前国内尚无存在的报道。PCR技术是一种快速灵敏的植物病毒检测方法,但核酸内的聚合酶抑制物会导致漏检现象,而只通过凝胶电泳进行结果判定会出现假阳性,这两方面限制了PCR技术在对外检疫中的应用。利用共价结合在PCR管壁上的引物特异性杂交诱捕核酸粗提液中的靶标核酸,洗掉杂质及抑制物质,在同一管内作RTPCR,凝胶电泳检测液相产物的同时对固相产物进行杂交检测,提高了结果的可靠性及灵敏度。利用所建立的HCRTPCRELISA成功地从法国进口葡萄苗中检出ToRSV。本方法还可用于其他植物病毒及转基因产品的检测。     

转基因植物检测技术的研究进展

现代植物基因工程使转基因植物及其产品越来越多地进入人们的生活,转基因植物安全性在世界范围内引起了广泛关注,对转基因植物检测技术的需求也越来越紧迫。就转基因植物检测技术的研究进展进行综述,重点介绍以基因和蛋白为目标的检测技术,包括PCR、ELISA和基因芯片技术的最新进展,并对不同方法的优缺点进行对比。此外,提出对特定代谢产物的检测是转基因植物检测的重要组成部分,是以后检测技术的发展趋势之一。最后,以差异蛋白为检测目标,结合研究工作提出基于双向电泳技术的转基因植物检测方法及其产品溯源方案。     

转基因产品成分检测技术研究进展

近年来随着转基因作物的大规模商业化种植以及转基因研发技术的不断发展,抗病、抗虫、耐除草剂、抗逆境和高产优质等转基因产品越来越多,应用也越来越广泛。转基因产品给人们带来便利和经济利益的同时,也带来了安全性问题的争议。为加强对转基因产品的管理,发展转基因产品成分检测技术尤为重要。目前,转基因产品的检测技术主要分为两类:一是基于外源核酸的检测技术,主要包括定性PCR技术、定量PCR技术、等温扩增技术和基因芯片技术等;二是基于外源蛋白的检测技术,主要包括酶联免疫吸附技术、Western blot检测技术和试纸条技术等。每种检测技术都有其优缺点和适用范围,在实际检测过程中,应根据检测的需要并结合转基因产品的类型和特点,选择最有效的检测技术或组合来满足检测的目的。就两类方法中主要检测技术的原理、优缺点和研究进展进行了简要概述,并就转基因检测技术的发展方向进行了总结和展望,旨在促进我国转基因检测技术更好地适应当前转基因领域的发展。     

转基因作物及加工品检测技术概述

随着公众对转基因产品关注度的提高和转基因标识制度的建立,快速、准确、灵敏地检测出转基因成分是客观发展的必然要求,建立高效、高通量的转基因检测技术已成为国内外的热点课题。我们简要介绍了转基因检测技术的概念、原理和研究进展,比较了各种转基因检测技术的优缺点,对转基因检测技术的发展方向进行了总结和展望。     

中国转基因大豆的产业化策略

大豆是事关人民生活和经济社会发展的重要农产品之一,提高大豆生产水平和增加自给能力,是中国农业生产必须解决的重大问题。由于中国耕地资源不足的限制,科技创新是提升大豆生产能力的唯一出路。转基因育种是推动大豆生产发展的颠覆性技术,对美国、巴西和阿根廷等世界主产国大豆产业的发展发挥了重要作用。经过20多年的科技创新,中国转基因耐除草剂和抗虫育种技术已经成熟,这些产品的产业化种植可显著降低大豆生产成本和提升单产水平。基于中国转基因大豆技术发展进度和大豆生产的国情特点,我们提出了采用如下策略科学有序推进产业化工作。一是,在产品应用时间上,按照单一耐草甘膦除草剂、多个基因耐草甘膦和草铵膦等多种除草剂,以及耐除草剂与抗虫等复合性状等产品,依次推进相关种子的产业化;二是,在产品区域布局上,按照靶标杂草和害虫的地理分布特点顶层设计各种耐除草剂和抗虫大豆产品的种植区域;三是,在生物安全管理上,研发应用抗性杂草和害虫种群监测与治理技术,延长转基因产品的使用寿命。同时,还要加强野生大豆资源的保护工作,降低转基因大豆基因漂移对野生大豆生物多样性的影响。     

MDA技术在转基因检测中的研究与应用

PCR技术是转基因植物及其产品检测的主要方法,但是因为其对模板纯度和质量有较高要求,对于低质量模板难以获得稳定的检测结果。本研究利用MDA技术可将低至10 copies的微量DNA样品扩增至可用PCR方法稳定检出;进一步利用MDA技术对转基因玉米有证标准物质Bt11粉末的单颗粒痕量样品释出DNA进行扩增,结合PCR扩增检测,玉米粉末颗粒中内源参照基因的检出率达到70%以上,转基因特异性序列检出与内源参照基因检出的比值,与标准物质标称值基本相符,为建立基于MDA技术转基因植物及其产品粉末颗粒等痕量样品检测方法奠定了基础,同时也为加工产品中复合性状转基因作物的检测提供了潜在的解决方案。     

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。     

转基因植物食品检测技术研究进展

随着转基因植物的迅速发展,转基因植物（GMO）含量大量涌向市场。出于对人类健康的考虑,GMO食品标识制度在世界范围内受到人们普遍关注。不论是对GMO食品贴标签,或是对GMO与非GMO原料的分别输送,GMO原料和食品的检测技术是必不可少的。GMO食品的检测主要有两种方法：一种是以DNA为基础的PCR法,另一种是从蛋白质水平出的ELISA法。比较全面地阐述了这两种检测方法的应用状况,重点介绍了PCR检测法及影响PCR检测方法的因素,作者预测DNA芯片检测法将是未来的发展方向。     

The development and standardization of testing methods for genetically modified organisms and their derived products

As the worldwide commercialization of genetically modified organisms (GMOs) increases and consumers concern the safety of GMOs, many countries and regions are issuing labeling regulations on GMOs and their products. Analytical methods and their standardization for GM ingredients in foods and feed are essential for the implementation of labeling regulations. To date, the GMO testing methods are mainly based on the inserted DNA sequences and newly produced proteins in GMOs. This paper presents an overview of GMO testing methods as well as their standardization.     

GMDD: a database of GMO detection methods

Background  

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.     

浅谈多国转基因产品标识制度对我国的启示

转基因产品（genetically modified organis, GMO）标识是为了表明该产品由转基因生物生产、加工而成的特殊标识,即标识产品中含有转基因成分。21世纪以来,全球共有60多个国家种植转基因作物,随之出现大量转基因产品。其标识问题关乎消费者的知情权和选择权而备受公众关注。一方面,随着全球转基因技术研发和应用的不断推进,国际上对转基因产品的标识管理更加关注与重视;另一方面,我国正处在有序推进生物育种产业化的关键期,转基因产品标识管理制度的与时俱进至关重要。2019年全球共有29个国家种植转基因作物,种植面积位列前十的国家依次是美国、巴西、阿根廷、加拿大、印度、巴拉圭、中国、南非、巴基斯坦以及玻利维亚,这10个国家的转基因作物总种植面积占全球总种植面积的97.9%。以其他9个国家的转基因标识管理制度为切入点,分析不同国家及不同标识类别的特点,旨在为我国的转基因产品标识管理工作提供启示与参考。     

A Microarray-based Detection System for Genetically Modified (GM) Food Ingredients

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO. Serge Leimanis, Marta Hernández, Sophie Fernández: These authors contributed equally to this paper.     

基于PCR技术的DNA分析测试关键要素

大多数国家对转基因生物研究与产业化日趋积极,把发展生物技术作为支撑发展、引领未来的战略选择,力求抢占新一轮经济和科技革命的先机与制高点。随之而来的是公众对转基因产品安全性的关注。转基因生物成分分析作为安全性评价的重要内容,发挥着举足轻重的作用,而基于PCR技术的转基因产品核酸成分分析是最经典、最广泛使用的方法。虽然此方法已普遍应用,但国内外鲜有较为全面的阐述此类分析测试要素的报道。为此,本综述对国内外相关方法的文献资料进行了梳理、比对和研究,结合已有的分析测试实验室的经验,提出了PCR测试体系的关键要素主要是标准和指南、方法的确认、标准物质及能力验证等四个方面,且在文中简明扼要的论述了各个部分的要点,供转基因核酸成分分析及相关领域工作者参考。     

Relative quantification in seed GMO analysis: state of art and bottlenecks

Reliable quantitative methods are needed to comply with current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) and GMO-derived food and feed products with a minimum GMO content of 0.9 %. The implementation of EU Commission Recommendation 2004/787/EC on technical guidance for sampling and detection which meant as a helpful tool for the practical implementation of EC Regulation 1830/2003, which states that “the results of quantitative analysis should be expressed as the number of target DNA sequences per target taxon specific sequences calculated in terms of haploid genomes”. This has led to an intense debate on the type of calibrator best suitable for GMO quantification. The main question addressed in this review is whether reference materials and calibrators should be matrix based or whether pure DNA analytes should be used for relative quantification in GMO analysis. The state of the art, including the advantages and drawbacks, of using DNA plasmid (compared to genomic DNA reference materials) as calibrators, is widely described. In addition, the influence of the genetic structure of seeds on real-time PCR quantitative results obtained for seed lots is discussed. The specific composition of a seed kernel, the mode of inheritance, and the ploidy level ensure that there is discordance between a GMO % expressed as a haploid genome equivalent and a GMO % based on numbers of seeds. This means that a threshold fixed as a percentage of seeds cannot be used as such for RT-PCR. All critical points that affect the expression of the GMO content in seeds are discussed in this paper.     

Testing for genetically modified organisms (GMOs): Past, present and future perspectives

This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve in the next decade. Challenges and limitations for the application of the test methods as well as to the interpretation of results produced with the methods are highlighted and discussed, bearing in mind the various interests and competences of the involved stakeholders. To better understand the suitability and limitations of detection methodologies the evolution of transformation processes for creation of GMOs is briefly reviewed.     

Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence

To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (<25%) of the GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.