应用单管半巢式PCR技术筛查转基因食品

建立转基因作物中常见的两种外源调控元件的单管半巢式聚合酶链式反应（polymerase chain reaction,PCR）测方法,为转基因食品的筛选检测提供一种快速、精确的方法。针对6 种转基因作物中CaMV35S启动子和NOS终止子的一致性核苷酸序列,分别设计巢式PCR的内、外引物,在测试不同引物组合的扩增效率基础上,建立CaMV35S启动子和NOS终止子的单管半巢式PCR方法。结果表明,上述方法对两种转基因成分具有良好的特异性,检测灵敏度分别为0.01%和0.05%,显著优于常规PCR方法。该方法具有便捷、准确、灵敏等特点,适合筛查食品中的转基因成分。     

多重PCR方法检测食品中转基因成分

根据转基因农作物中最常用的花椰菜花叶病毒启动子 (CaMV 3 5S)和根癌农杆菌终止子(NOS)的序列 ,设计合成了两对不同的引物和相对应的两种荧光双链探针 (FDCP) ,分别建立了多重PCR、应用FDCP的实时荧光PCR同时检测转基因成分 3 5S启动子和NOS终止子的方法 .并利用该套方法对马铃薯、大豆、玉米、甜椒、番茄等实物样品进行了检测 ,发现 1 3份样品中有 6份检出3 5S启动子、NOS终止子 ,其余 7份样品的检测结果为阴性 .表明作者建立的多重PCR方法能有效检测出 3 5S和NOS成分 ,其中多重PCR法具有灵敏度高、特异性好的特点 ,多重荧光PCR法则更为简便、快速、准确     

Quantitative detection of the 35S promoter and the NOS terminator using quantitative competitive PCR

 DNA-based analytical methods are often used to verify the presence of genetically modified organisms (GMOs) in food. In Switzerland, a preliminary study, organized by a subcommission of the Swiss Food Manual, of different polymerase chain reaction (PCR) systems for the detection of GMOs showed that the application of qualitative PCR systems can lead to interlaboratory differences of at least a factor of 10. These differences can be diminished using internal standards (competitors). The quantitative competitive (QC) PCR for the detection of the 35S promoter or the NOS terminator in food samples is presented. The GMO content of food samples can be determined using QC-PCR. Received: 2 July 1998 / Revised version: 15 October 1998     

Detection of Residues of Genetically Modified Soybeans in Breaded Fried Turkey Cutlets

The presence of genetically modified organisms (GMOs) in food products is usually ascertained by the polymerase chain reaction (PCR) or nested PCR if sensitivity has to be increased. Since most, if not all, GMO products are of plant origin, the target sequences are the 35S promoter or NOS terminator. The extreme sensitivity of nested PCR can be misleading if the results are not interpreted correctly, since contamination of non-GMO products with residual amounts of GMO may be positive. We report that breaded turkey breast cutlets labeled as containing GMO soybean products were actually prepared from wheat flour that had been contaminated with transgenic soybean.     

Development of a quadruplex-real-time-PCR for screening food for genetically modified organisms

With the ever increasing number of genetically modified plants authorized worldwide, including in the European Union, high throughput detection methods need to be developed. In this paper, a quadruplex-real-time-PCR method is described which allows rapid and simultaneous screening of food for the presence of target DNA sequences from the cauliflower mosaic virus 35S promoter, the NOS terminator from Agrobacterium tumefaciens, the soya reference lectin gene and the maize reference alcool dehydrogenase gene (adh). Three of the four primers and probe combinations have already been published elsewhere, whereas primers and probe for NOS terminator-PCR were developed in-house. Validation data show sensitivities down to five copies for 35S promoter and NOS terminator PCR, even when target sequences of the competing PCRs are in large excess. Thorough adjustment of primer and probe concentrations allowed high individual PCR efficiencies with negligible physical cross-talk between the four detection channels. This method provides a basis for a rapid screening of food for the most frequently used regulatory elements present in GM crops authorized for food in the European Union. In addition it provides information about the presence of species which are possibly genetically modified.     

肉制品中植源性转基因成分多重荧光定量PCR检测方法的建立

为改善产品品质,肉制品加工过程中常常添加植物源性成分,当前转基因农作物的商品化及其在市场上 的广泛流通导致肉制品中被带入植源性转基因成分的风险增加。以转基因植物中常涉及的调控元件CaMV 35S启 动子、NOS终止子以及标记基因NPTⅡ为检测目标,设计相应的引物和Taq man探针,利用载体pRⅠ 101-AN DNA 为模板,通过优化反应体系和反应参数,建立肉制品中植源性转基因成分的单重和多重荧光定量聚合酶链式反应 （polymerase chain reaction,PCR）检测方法。通过比较分析,考察多重荧光定量PCR检测方法的灵敏性、重复性和准 确性,结果表明,多重荧光定量PCR检测方法灵敏度高、重复性好且与单重体系的检测结果具有很好的一致性。     

转基因大豆的PCR-免疫层析筛查方法研究初探

目的建立转基因大豆的PCR-免疫层析（PCR—ICT）快速筛查新技术。方法利用转基因植物通常含有的CaMV35S启动子作为转基因成分的筛查标记，根据其序列设计特异性引物和探针，分剐用生物素和地高辛标记，用胶体金免疫层析技术检测并鉴定PCR产物。结果用新建立的PCR—ICT方法可以检出含0．5％转基因大豆的标准品，对大豆样品的检测结果与琼脂糖凝胶电泳检测结果一致。结论PCR—ICT方法通过DNA杂交和金标显色来同时检测、鉴定PCR产物，可以简便、快速地筛查转基因产品。     

PCR方法对转基因食品定性检测的研究

转基因食品的检测一般基于聚合酶链式反应（PCR）方法，对35S启动子和NOS终止子进行筛选。本实验以转基因大豆、玉米（购自Fluka）为参照物，转基因含量为0％，1％，2％，5％的样品均获得正确识别。以上介绍的方法能对转基因原材料及加工成品实施高精度、高灵敏的检测。     

Detection of cauliflower mosaic virus by the polymerase chain reaction: testing of food components for false-positive 35S-promoter screening results

 Today DNA-based techniques are very common for the detection of genetically modified organisms (GMOs) in food products. For fast and easy detection of GMOs, polymerase chain reaction (PCR) screening methods, which amplify common transgenic elements, are applied in routine analysis. These techniques do not allow differentiation between GMOs and the natural occurrence of transgenic elements, such as the 35S-promoter of cauliflower mosaic virus (CaMV) or the NOS-terminator of Agrobacterium tumefaciens, and thus may result in false-positive detection of GMOs. In this study we evaluated three different existing 35S screening systems and report the development of two new CaMV-specific PCR systems. These PCR systems based on CaMV-specific genes allow the identification of positively screened 35S food samples as naturally virus-infected products or plants. Seven food samples tested positive in routine 35S screening analysis and negative in GMO specific systems were investigated using the new virus-specific PCR systems. In all seven samples CaMV was detected. Received: 26 April 1999 / Revised version: 28 June 1999     

主要农作物转基因成分检测能力验证结果与分析

目的 提升实验室主要农作物转基因成分检测能力水平,积累检测工作经验,做好主要农作物监管检测工作的准备。方法 根据组织者提供的能力验证作业指导书和相关农业农村部公告,对玉米、大豆、水稻、油菜四种主要农作物的20种样品进行了转基因成分检测。结果 20种样品中,CaMV 35S启动子、NOS终止子和相应的内标准基因的能力验证检测均获得满意结果。结论 实验室通过普通PCR和实时荧光PCR的方法均获得一致结果,证明实验室熟练掌握了相关标准和检测方法,具备开展主要农作物转基因成分检测的能力。     

花椰菜花叶病毒CaMV 35S启动子LAMP检测方法的建立

针对花椰菜花叶病毒(CaMV)35S启动子的保守序列设计特异性引物,并进行条件优化,建立CaMV35S启动子的LAMP检测方法。该方法具有良好的特异性,检测灵敏度可达0.002%。对80份各类植物及其加工品进行检测,检测结果与实时荧光PCR法完全相符。CaMV35S启动子的LAMP检测方法,在检验检疫行业和食品加工业具有广阔的应用前景,对于加强转基因产品检测监管,促进进出口贸易具有重要意义。     

PCR法检测大豆加工食品中的转基因成分

通过分子生物学手段,以聚合酶链式反应(PCR)技术为基础,建立了检测大豆加工食品中转基因成分的方法。实验分别采用CTAB法和试剂盒(Kit)法对大豆锅巴、豆浆、豆奶粉、豆腐、豆腐丝等五种大豆加工食品中的DNA进行了提取,用内标基因Lectin对此两种方法的提取效果进行了比较,并以提取的DNA为模板,利用不同的引物分别对目标基因35S和NOS进行了PCR扩增和琼脂糖凝胶电泳检测。结果表明:Kit法的DNA提取效果优于改良CTAB法,上述五种大豆加工食品中均检测出35S启动子和NOS终止子,且均含有转基因成分。     

转基因大豆及其制品二重荧光定量PCR的建立与验证

研究选用花椰菜花叶病毒35S启动子(P-35S)和根癌农杆菌胭脂碱合成酶基因终止子(T-NOS)为靶标基因,运用多重实时荧光PCR技术对转基因大豆及其制品进行快速筛选检测。实验通过样品核酸提取与质控,多重引物及荧光探针的设计与筛选,反应条件和反应体系的对比优化,摸索出二重荧光定量PCR检测的最佳反应体系。同时通过特异性、重复性、灵敏性和适用性实验验证,确保了该方法在同时检测2个靶标基因时,无荧光信号的相互干扰,不会出现假阴性和假阳性结果。结果表明,方法特异性高,可同时筛查两个靶标基因,扩增效率在90%~110%之间,标准曲线决定系数R2>0.98,确定了最低检测限为2拷贝/μL。开发和建立的二重荧光定量PCR检测技术可以实现一管多检的实际需要,降低试剂成本,缩短检测时间,为大豆及其深加工产品转基因成分的快速检测提供了有效方法,为促进食品进出口提供技术保障。     

转基因大豆DNA检测芯片的研究

为提高对转基因大豆的监督检测能力，研制了转基因大豆DNA检测芯片。根据转基因大豆(Roundup Ready)中所转入的外源基因，选择CaMV35S启动子、NOS终止子、NOSIEPSPE基因和内源Lectin基因设计特异性引物，采用多重PCR法对待测样品进行扩增，通过缺口平移法合成DIGdUTP标记杂交探针，并制备基因芯片。在对PCR反应和扩增产物与芯片杂交条件进行优化的同时，比较了芯片检测的特异性和重复性，并对检测的灵敏度进行测试。结果表明，该方法具有较好的特异性和重复性，检测灵敏度可达0．5％，由于采用了多重PCR技术，一次可同时检测多个基因，提高了检测的准确性和效率。     

Combinatory SYBR® Green Real-Time PCR Screening Approach for Tracing Materials Derived from Genetically Modified Rice

Two real-time PCR approaches for the detection of genetically modified (GM) rice were tested: (1) a combination of SYBR® Green real-time PCR methods detecting the 35S promoter (P-35S) of Cauliflower Mosaic Virus, the nopaline synthase terminator (T-nos) of Agrobacterium tumefaciens and the Bacillus thuringiensis (Bt) CryIAb/Ac toxins and (2) a P-35S/T-nos duplex TaqMan® real-time PCR system. Both systems correctly recognized their respective targets in control samples of Bt63, Kefeng6 and KMD1 insect-resistant and LLRice62 and LLRice601 herbicide-resistant rice. Due to its lesser specificity but broader genetically modified organism (GMO) coverage capacity, the SYBR® Green real-time PCR approach was further tested in more detail. Melting curve, capillary and agarose gel electrophoresis analyses indicated that the P-35S, T-nos and CryIAb/Ac targets in the GM rice events are similar to the corresponding targets present in many known GMOs. High-resolution melting analysis showed that the CryIAb/Ac targets of the GM rice events Bt63 and Kefeng6 matched best the corresponding Bt11 CryIAb sequence. Digital PCR analysis on genomic DNA from the GM rice Bt63 and Kefeng6 events indicated that both GMO contained multiple inserts. Sensitivity tests showed that all SYBR® Green real-time PCR methods could detect their targets at less than an estimated five copies per reaction. Finally, it was shown that these SYBR® Green real-time PCR methods could detect low levels of their targets in rice consignments originating from China. Together, these results demonstrated that a ‘P-35S and T-nos and CryIAb/Ac’ combinatory SYBR® Green real-time PCR screening is highly suited to trace the respective targets including the possible presence of Bt63, Kefeng6 and KMD1 GM rice materials in food products.     

应用依赖解旋酶DNA扩增技术检测转基因大豆

目的：基于依赖解旋酶DNA恒温扩增（helicase-dependent isothermal DNA amplification,HDA）技术,建立快速检测转基因大豆的方法。方法：采用以CaMV35S、NOS、CP4-EPSPS 3 种外源基因和内源基因Lectin（大豆凝集素基因）为目的片段设计4 对特异引物,建立反应体系,通过HDA方法对内源基因和3 种外源基因的检测特异性和灵敏度进行实验,并对结果进行同源性分析。结果：建立了转基因大豆HDA检测法,检测特异性良好,3 种外源基因的检出限为0.2%。结论：转基因大豆的HDA检测方法具有普通聚合酶链式反应的特异、灵敏等特点,并且对仪器要求更低,极适合基层实验室使用,具有广阔的应用前景。     

广州市售豆制品转基因成分的检测与分析

为了解广州市市售豆制品的转基因情况,对广州市售散装豆制品和预包装豆制品分别进行抽样检验.采用改良十六烷基三甲基溴化铵（CTAB）法提取大豆DNA,利用核酸定性PCR、实时荧光PCR及环介导等温扩增技术（LAMP）对外源基因CaMV5S,NOS和EPSPS进行检测.调查共抽检豆制品207份,研究结果表明,在检测的159份散装豆制品中,87份检出转基因成分;在48份预包装豆制品中,18份检出转基因成分.散装豆制品无任何食品标签,而预包装豆制品均无转基因食品标识.     

Detection and identification of genetically modified EE‐1 brinjal (Solanum melongena) by single,multiplex and SYBR® real‐time PCR

BACKGROUND: Brinjal is an important vegetable crop. Major crop loss of brinjal is due to insect attack. Insect‐resistant EE‐1 brinjal has been developed and is awaiting approval for commercial release. Consumer health concerns and implementation of international labelling legislation demand reliable analytical detection methods for genetically modified (GM) varieties. RESULTS: End‐point and real‐time polymerase chain reaction (PCR) methods were used to detect EE‐1 brinjal. In end‐point PCR, primer pairs specific to 35S CaMV promoter, NOS terminator and nptII gene common to other GM crops were used. Based on the revealed 3′ transgene integration sequence, primers specific for the event EE‐1 brinjal were designed. These primers were used for end‐point single, multiplex and SYBR‐based real‐time PCR. End‐point single PCR showed that the designed primers were highly specific to event EE‐1 with a sensitivity of 20 pg of genomic DNA, corresponding to 20 copies of haploid EE‐1 brinjal genomic DNA. The limits of detection and quantification for SYBR‐based real‐time PCR assay were 10 and 100 copies respectively. CONCLUSION: The prior development of detection methods for this important vegetable crop will facilitate compliance with any forthcoming labelling regulations. Copyright © 2012 Society of Chemical Industry     

利用实时荧光PCR方法检测转Bt基因大米

应用实时荧光PCR技术对转基因大米进行了定性和定量检测研究.本研究以转基因B163大米为材料,采用TaqMan探针技术,对大米中的内源基因蔗糖磷酸合酶SPS和转基因水稻中普遍存在的外源基因CaMV35S启动子、NOS终止子以及苏云金芽孢杆菌(Bacillusthndngiemis,简写为Bt)杀虫晶体蛋白基因Cry1Ac进行了实时荧光PCR研究,并对外源基因Cry1Ac进行了定量检测和敏感性分析.该实时荧光PCR方法检测结果和常规PCR结果一致,同时不用进行凝胶电泳,更为快速、简便,降低了污染机会,可用于转Bt基因大米的定性和定量检测.     

Development of 10 new screening PCR assays for GMO detection targeting promoters (pFMV, pNOS, pSSuAra, pTA29, pUbi, pRice actin) and terminators (t35S, tE9, tOCS, tg7)

p35S promoter and tNOS terminator are the two primary targets for genetically modified organism (GMO) screening. An increasing number of genetic constructions do not contain p35S and tNOS elements; therefore, new screening assays are required. The use of a larger number of screening methods provides a better coverage of the EU-unapproved GMOs and is a cost-effective approach due to the decrease of tests required for identification. In the present study, new real-time PCR screening assays were developed targeting 10 promoter and terminator elements used in genetically modified constructs: pFMV, pNOS, pSSuAra, pTa29, pUbi, pRice actin, t35S, tE9, tOCS, and tg7. Specificity was verified against different plant species, and the limit of detection was determined on plasmid and genomic reference materials. Criteria of performance were successfully tested taking into account the recommendations of international guidelines. It means that these assays can be considered as ready for an inter-laboratory validation.