应用PCR-ELISA技术检测转基因产品的研究

建立并优化了转基因大豆与玉米的DNA提取,CaMV35S启动子和T－NOS终止子的序列特点设计特异性引物与探针，应用PCR－ELISA检测技术，建立了转基因大豆与玉米中常用外源基因的快速检测体系，并应用于进出境产品的转基因检测实际工作中。结果表明，建立的PCR－ELISA法具有操作简便，灵敏特异，结果准确的，可对转基因大豆，玉米及其它转基因产品进行定性和定量检测。     

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。     

Screening food products for the presence of CaMV 35S promoter and NOS 3′ terminator

Biotechnology has enabled the modification of agricultural materials in a very precise way, thereby improving productivity and yields of economically important crops. There are a number of methods available for detecting genetically modified organisms (GMOs). In the present investigation, a qualitative PCR technique has been adopted in order to discriminate between genetically modified and non‐modified food products. The qualitative PCR assay employs primers specific for genetic elements that are used to generate genetically engineered agricultural crops. Two of the most common primers used for the detection of GMOs, 35S promoter and NOS 3′ terminator, have been tested over a panel of 24 food products purchased from the local market. The results indicated that, out of the 24 food products tested, three products gave positive results with the 35S promoter. The NOS 3′ primers gave negative results with all tested samples. Copyright © 2005 Society of Chemical Industry     

Establishment of a quadruplex real-time PCR for screening of genetically modified tomatoes

With the increase in number of the genetically modified (GM) crops authorized worldwide and specific labeling legislation established by many countries, a reliable and efficient method for routine screening of raw material or processed food products needs to be developed. In this paper, a quadruplex quantitative real-time PCR (qPCR) system is described which allows simultaneous detection of one tomato endogenous gene and three most frequently used transgenic elements in GM products: cauliflower mosaic virus 35S promoter, Agrobacterium tumefaciens nopaline synthase terminator, and neomycin phosphotransferase II gene. The specificities of the assays are optimized and validated. In the quadruplex qPCR system, the detection ranges for all of the four genes were determined to be 8–80,000 copies per reaction. Finally, the established detection system was applied in amplification of exogenous and endogenous genes from 33 raw materials and 35 processed products samples. The results indicate that quadruplex qPCR method is feasible for screening of GM tomato products, even for some processed food. As this detection system could be easily applied to the detection of transgenic elements in other plant species, we expect it will meet the challenges of routine GM crop detection resulting from a rapid increase in the number of GM crops in the future.     

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     

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。     

微滴式数字PCR和实时荧光PCR在豆奶饮料转基因成分检测中的应用

为探索数字PCR在转基因成分筛选检测中的应用,解决现行转基因食品标准实施中的问题,完善我国转基因食品的检测监管体系。本文选取转基因启动子Ca MV35s和终止子NOS基因为靶标,大豆内源基因Lectin为内标,以转基因大豆标准品分别测定数字PCR和实时荧光PCR的浓度和含量检测低限,并应用于30批次豆奶饮料转基因成分实测。结果表明,数字PCR在转基因筛查的浓度检测低限达到0.04ng/反应,含量检测低限达到0.05%,均优于实时荧光PCR(0.2ng/反应,1%),且能够满足最严欧盟转基因标识阈值0.9%的检测要求。在豆奶饮料实际应用中发现,26批次样品两个平台检测结果一致为阴性,1批次一致为阳性,3批次Ct值在34.59～38.28之间的样品经数字PCR检测得到确切结果,说明数字PCR可辅助确认RT-PCR可疑结果,解决现行标准判定难题。     

Two quantitative multiplex real-time PCR systems for the efficient GMO screening of food products

According to the EU and Swiss food legislation, only deregulated traits of transgenic plants are allowed to be imported and sold to the consumer. In order to control imports of soya and maize products from retailers, efficient and reliable methods for the detection and quantification are a prerequisite. The screening for specific DNA elements characteristic of transgenic plants is crucial for further analysis and has a major impact on the efficiency of the whole analysis workflow. To allow laboratories to efficiently and reliably screen food products for transgenic plant products, two novel multiplex real-time polymerase chain reaction (PCR) systems were developed and validated. One system determines DNA contents from maize, soya, cauliflower mosaic virus (CaMV) 35S promoter (P35S), NOS terminator from Agrobacterium tumefaciens and CaMV, and the second PCR system simultaneously detects DNA sequences from figwort mosaic virus promoter (PFMV), from bar gene of Streptomyces hygroscopicus, from gene coding for phosphinothricin acetyltransferase (PAT) and from a DNA construct of enolpyruvyl shikimate phosphate synthase gene (CP4-EPSPS) and Arabidopsis thaliana (CPT2). The tests exhibit good specificity and a limit of detection of at least 0.1 % for all analytes.     

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     

转基因"华番一号"番茄的筛选和特异性的定性、定量PCR检测方法

为给转基因植物监测提供技术支持，建立了转基因“华番一号”番茄筛选和特异性的定性、定量PCR检测方法。转基因“华番一号”的筛选PCR检测主要以转基因通用元件CaMV35S启动子和NOS终止子为目的基因片段，特异性PCR检测以转基因外源重组子的CaMV35S启动子和反义EFE基因的相邻序列为目的片段；实验同时设立番茄的LAT52基因为转基因番茄定性、定量PCR检测的内对照基因。在所建立的PCR检测体系中，定性PCR筛选和特异性检测的检测极限为68个拷贝，实时定量PCR方法的检测极限为3个拷贝；筛选定量．PCR检测的定量极限为3个拷贝，特异性定量PCR检测的定量极限为25个拷贝。最后通过对2个已知含量的转基因番茄“华番一号”混合试样的检测，证明了该体系可以有效地用于转基因番茄“华番一号”的筛选和特异性的定性、定量PCR检测。     

Inter-laboratory Testing of GMO Detection by Combinatory SYBR®Green PCR Screening (CoSYPS)

Combinatory SYBR®Green real-time PCR Screening (CoSYPS) is an efficient, sensitive approach for detecting complex targets such as genetically modified organisms (GMOs) in food and feed products. GMO analysis for legal purposes has become increasingly complex and costly due to the diversity in recombinant targets present in the different GMOs. For this reason, screening for the presence of GMOs is in general the first step in the detection of GM material in a product. CoSYPS allows detecting the large majority of globally commercial GMOs using SYBR®Green real-time PCR methods for six GM targets (P35S, Tnos, CryIAb, CP4-EPSPS, PAT and BAR) combined with species-specific PCR methods (e.g., maize, soy, rapeseed). Here, the results of an inter-laboratory trial on seven samples with different GMO mixtures at different levels are presented. In total, 13 laboratories participated in the trial and the currently most frequently used PCR analysis platforms are represented. The inter-laboratory study clearly demonstrates that PCR methods used in CoSYPS form a very robust GMO screening system. Sensitivity, specificity, positive and negative predictive values are for all PCR methods higher than 95 % for all samples. Together, these results show that the SYBR®Green real-time PCR methods used in CoSYPS are effectively applicable to different PCR platforms and amendable to configuration into a sensitive high-throughput GMO screening and decision support tool.     

大豆加工食品中转基因成分多重PCR检测体系的建立

以大豆内源基因 (Lectin)、筛选基因 3 5S启动子 (Cauliflowermosaicvirus 3 5S ,CaMV3 5S)、Nos终止子 (Nopalinesynthase,Nos)和外源基因 (5 enolpyruvylshikimate 3 phosphatesynthase ,Ep sps)为检测对象 ,通过对PCR扩增体系中各引物终浓度及PCR扩增过程中退火温度的探讨 ,研究了不同引物终浓度配比及退火温度对转基因大豆多重PCR检测的影响 ,建立了大豆加工食品中转基因成分多重PCR检测体系。结果表明 ,当各组引物的终浓度分别为 1 0、2 0、2 0、3 0 μmol/L即引物终浓度配比为 1∶2∶2∶3 ,退火温度为 5 5 4℃时 ,所建立的多重PCR检测方法能够有效地检测出大豆中的转基因成分 ,具有特异性好 ,简便 ,快速 ,准确等优点。     

Development of two screening duplex PCR assays for genetically modified organism quantification using multiplex real-time PCR master mixes

Since 2001, the traceability and labelling of genetically modified organism (GMO) food and feed derived products are obligatory in the European Union. Genetically modified organisms (GMO) are commonly detected via PCR tests. These tests typically involve several steps: (1) screening (2) construct specific (3) event specific and (4) reference gene. Screening tests are based on sequences frequently used for GM development, allowing for the detection of a large number of GMOs. To improve GMO detection efficiency, using specific multiplex master mixes, we developed two real-time PCR screening duplex PCR assays for the detection of P35S/Tnos and Pnos/T35S sequences. By combining these tests, we were able to reduce the time and cost of analysis. For the Pnos/T35S duplex, good sensitivity was obtained using one of the mixes compared to the others. Both duplexes had 100% specificity when tested on DNA from GM maize, rapeseed and soybean. When the duplexes were tested on DNA containing various amounts of GM maize and soybean, the corresponding targets were detected. The detection limit of our methods was found to be between 2 and 8 haploid genome copies for both P35S/Tnos and Pnos/T35S tests. In summary, with high efficiency and good linearity, the proposed two screening duplexes allow for more efficient GMO detection.     

多重实时荧光PCR快速检测转基因大豆及其加工产品

本研究运用多重实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因大豆及其深加工制品进行筛选检测。通过设计大豆内源基因植物凝集素(Lectin)和常用的外源基因花椰菜花叶病毒35S启动子(CaMV35S)、根癌农杆菌胭脂碱合成酶基因终止(nos)的特异性引物和探针,反应条件和反应体系的优化,特异性、重复性和灵敏性的实验比对分析等开发建立了多重荧光定量PCR检测技术。以10%Roundup Ready转基因大豆标准品为材料,建立并优化转基因大豆的定量检测体系,对大豆中的转基因成分进行定量分析。结果表明:该方法重复性好,检测特异性强,扩增效率在90%～110%,标准曲线相关系数R2≥0. 98,确定了最低检测限为每20μL反应2. 4个拷贝。结论:由于使用多重实时荧光PCR技术,可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,为促进农产品和食品进出口提供技术保障。     

Screening and construct‐specific detection methods of transgenic Huafan No 1 tomato by conventional and real‐time PCR

Genetically modified (GM) tomatoes have been approved for commercialization in many countries since the first GM tomato FLAVR SAVR was permitted for planting in 1994. In China, GM tomato Huafan No 1 with a character of long shelf‐life was the first GM plant which was approved for commercialization in 1996. To meet the requirement of the GM tomatoes labeling policy that has been actualized in China since 2001, screening and construct‐specific PCR detection methods for detecting the universal elements transformed into tomato, such as cauliflower mosaic virus 35S (CaMV35s) promoter and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, and the specifically inserted heterologous DNA sequence between CaMV35s promoter and anti‐sense ethylene‐forming enzyme (EFE) gene were set up. To make the detection methods normative, a novel single copy tomato gene LAT52 was also used as an endogenous reference gene in the PCR detection systems. The limit of detection of screening and construct specific detection methods for Huafan No 1 was 68 haploid genome copies in conventional PCR detection, and three copies in TaqMan real‐time PCR detection. The limit of quantitation of screening quantitative PCR assays for Huafan No 1 was three copies and was 25 copies for construct‐specific quantitative PCR. Two samples with known Huafan No 1 tomato content were detected using the established conventional and real‐time PCR systems, and these results also indicated that the established Huafan No 1 screening and construct‐specific PCR detection systems were reliable, sensitive and accurate. Copyright © 2005 Society of Chemical Industry     

转基因稻米加工食品的PCR检测技术研究

本文对转基因稻米及其加工食品进行了针对标记基因bar、CaMV35S启动子和Nos终止子DNA序列的PCR检测.结果表明,改良SDS法适于提取转基因稻米以及经过蒸煮、微波膨化加工后稻米食品中的DNA并能够满足PCR检测的需要.食品加工使稻米基因组DNA降解为较小的片段,但不影响PCR检测效果,PCR技术能够检测稻米加工食品中的转基因成分.     

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。     

Development of a real‐time multiplex PCR system for the quantitative detection of Chinese GM rice

BACKGROUND: Rice is one of the most important staple food crops for more than half the global population, who rely on it for as much as 80% of their diet. By one estimate, the world population is projected to grow to approximately 11 billion people by the year 2050. So it is a formidable task to meet the future demand. For this reason, breeders make a great deal of effort to produce new rice varieties with traits such as higher yield, improved nutritional content and better resistance to disease and pests, via transgenic biotechnological protocols. Dozens of transgenic rice lines have been developed since the first transgenic rice plant production in the late 1980s. With the rapid approach of transgenic rice commercialisation, it is becoming necessary to develop techniques capable of detecting and quantifying genetically modified (GM) rice. RESULT: Here we describe a method in which transgenic DNA is quantified by amplifying part of the 35S‐CaMV promoter and standardising it against an amplified portion of an endogenous single copy, rice specific gene encoding sucrose phosphate synthase. Both reactions are performed simultaneously in a single tube. Standard calibration curves were developed by diluting DNA extracted from a blend of non‐transgenic (c.v., Nipponbare) and 5% KMD2 transgenic rice. The method was tested for the quantification of the five GM rice events, including KMD2, Wan 21A, GC‐1, H1597 and TR4, which contain the 35S‐CaMV promoter. The coefficient of variation varied from 3.15% to 12.84%, which is up to acceptance criterion over the dynamic range of the method. CONCLUSION: In this study, we successfully applied a multiplex real‐time PCR assay to GM rice, which employed SPS as the endogenous reference gene and the gene regulation element 35S‐CaMV promoter as a GMO marker. The detection limit and limit of quantification is sufficient to comply with all relevant regulations in the EU and worldwide. The detection system could be applied in routine analysis for the quantification of GM rice in food materials, such as instant rice, unpolished rice, rice flours, biscuit powders, and starch. It may prove useful with regard to a robust screening technique of broad utility as transgenic rice enters global commodity markets. Copyright © 2009 Society of Chemical Industry     

Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU and several other countries requires that foods containing genetically modified organisms (GMOs) should be approved and labelled. This has necessitated the development of methods for detection of such materials. For screening purposes these methods should preferably enable detection of several different GMOs. Here we present a simple, robust, qualitative, nineplex PCR method for event-specific detection of maize T25, GA21, TC1507, MON863, MON810, NK603, construct specific detection of BT176, BT11 and detection of the endogenous hmga maize reference gene. PCR is carried out with primers labelled with fluorescent groups and the amplicons are detected using fluorescence capillary electrophoresis. Using mixtures of DNA from different certified reference materials, the detection limit was determined to approximately 0.1% for each GMO. Good agreement was observed in 85 of 88 determinations when eleven food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. Discrepancies were only observed for one GMO at or close to the detection limit. The presented method is therefore suitable for screening purposes for food and feed containing the most common maize GMOs.     

大豆异黄酮中转基因大豆成分检测研究

利用含有35S启动子、NOS终止子片段的质粒,确定相应检测方法的灵敏度为15个拷贝和150个拷贝;采用扩增CaMV35S启动子的2对引物、扩增NOS终止子的2对引物、扩增EPSPS目的基因的1对引物,对大豆异黄酮样品中转基因成分进行了检测,结果均扩增出了特异性片段,且测序结果正确。