实时荧光定性聚合酶链式反应标准方法检测转基因产品的验证及应用

目的验证实验室大豆、玉米和水稻及其制品转基因检测方法,并应用于实际样品检测。方法根据GB 19495.4-2018《转基因产品检测实时荧光定性聚合酶链式反应(PCR)检测方法》要求对无转基因标识的样品进行转基因成分检测。结果方法验证满意。40批次样品(大豆、玉米和水稻及其制品)中发现1批次的转基因成分检出,检出率为2.5%。结论市场中绝大部分未标示转基因成分的大豆、玉米和水稻及其制品确实未检出转基因成分,仅有极少数产品含有转基因成分,但未进行有效标识。     

实时荧光PCR定量检测加工产品中转基因玉米Mon810成分

采用实时荧光PCR技术，建立了定量(性)鉴定检测加工产品中转基因玉米Mon810成分的方法。实验设计的可以扩增玉米自身基因和外源基因边界序列的引物和探针具有品种和品系特异性，特异性地检测出食品、饲料等加工产品中转基因玉米Mon810成分。某些检测样品不仅检出转基因玉米Mon810成分，还同时检出其它转基因玉米品系或其它转基因品种。本研究实验建立的转基因玉米Mon810品系鉴定检测方法，即可以用于加工产品中转基因成分的定量检测(检测低限为0．1％)，也可以用于定性检测，或作为常规PCR定性检测后的确证实验方法。     

转基因玉米59122品系的特异性检测

使用反向聚合酶链式反应(PCR)技术克隆了转基因玉米59122的外源基因与玉米基因组之间的两段侧翼序列,并据其左侧侧翼序列设计了具品系特异性的引物,运用半巢式PCR技术建立了59122的品系特异性二重PCR检测方法,扩增片段100bp,横跨pat终止子与转基因玉米侧翼基因之间。以转基因玉米59122、MON863、MON810、GA21、NK603,转基因大豆Roundup Ready和转基因油菜GT73等为材料,证明本方法与其他转基因作物具有高特异性。本方法在检测59122时,确定出连接体系中线性DNA的最佳质量浓度为1ng/μL左右,检出限达到0.1%,灵敏度为38个单倍体基因组拷贝数。因此可准确、快速、高效地检测转基因玉米及其产品,或作为常规PCR定性检测后的验证方法。     

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。     

大豆及其制品中转基因成分的二重PCR检测

为了建立能同时检测多种转基因成分的二重PCR检测方法,以转基因大豆(RoundupReady品种)为实验材料,优化了二重PCR的反应体系和反应条件,包括引物浓度、TaqDNA酶用量和退火温度等;比较了二重PCR和单一PCR的灵敏度和检测含量。结果表明:二重PCR的灵敏度和检测含量与单一PCR的相当;用建立的二重PCR方法从大豆、豆腐、豆粕和油炸豆腐中筛选出含转基因成分的阳性样品;二重PCR与单一PCR相比,具有节约试剂、省时等特点,在转基因产品检测上具有很好的推广价值。     

利用PCR方法检测转基因大豆加工食品中的修饰基因

市场上的转基因产品以及深加工产品越来越多,其安全性引起全世界的极大争论,很多国家的检验检疫机构相继开展了转基因产品的检测工作。本文以转基因大豆类食品为材料,以聚合酶链式反应(PCR)方法为基础,选择适用于转基因食品安全性检验的核酸检测技术,针对抗除草剂Round up Ready (RR)大豆的插入基因进行PCR检测,建立适合转基因大豆类食品的检测方法．该方法简便快速、检测结果与标准及其他文献资料相符．     

TaqMan探针用于转基因食品的荧光定量PCR检测

选择了内源基因大豆植物凝集素 (lectin)、玉米转化酶 (invertase)和外源基因抗草甘膦除草剂的 5 烯醇式丙酮酰莽草酸 3 磷酸合成酶 (cp4epsps)、抗欧洲玉米螟的苏云金芽孢杆菌杀虫毒蛋白 (cryIAb)的TaqMan探针 ,确定了探针浓度和镁离子浓度等反应条件 ,分别对转基因大豆和转基因玉米系列标准品进行内源基因和外源基因的荧光PCR扩增 ,在PCR反应过程中分别以TET和FAM两种荧光通道信号分别追踪同一样品DNA的内源基因和外源基因的扩增动力学变化 ,并依此绘制了ΔCt值与转基因食品百分比含量之间的标准曲线 ,建立了转基因大豆和转基因玉米的TaqMan -荧光定量PCR检测方法 ,该方法具有探针设计较简便、成本较低的特点 ,初步实现了对转基因食品的定量分析及品种鉴定。     

荧光PCR和数字PCR法检测转基因DAS-44406-6品系大豆

目的：建立实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测转基因DAS-44406-6品系大豆的定性检测方法和使用数字PCR检测转基因DAS-44406-6品系大豆的定量检测方法。方法：针对转基因DAS-44406-6大豆品系,进行5’-RACE,测定该品系转基因大豆外源片段与大豆染色体重组的边界序列,并根据该边界序列设计引物和探针。使用23 种非DAS-44406-6品系转基因植物作为阴性对照测试实时荧光PCR引物和探针的特异性,以DAS-44406-6品系样品制备6 个含量梯度的样品进行检测低限实验。使用数字PCR技术进行定量检测,并确定定量检测的低限。结果：建立的转基因DAS-44406-6大豆品系的实时荧光PCR特异性检测方法品系鉴定特异性较强,实时荧光PCR检测方法的检测低限在模板DNA浓度为100 ng/反应时,为0.01%的转基因大豆含量,约为16.6 个拷贝的DAS-44406-6基因组DNA;数字PCR检测方法的检测低限在模板DNA浓度为0.5 ng/反应、转基因大豆含量为1%时,相对标准偏差为0.7%。因此,建立的转基因DAS-44406-6大豆品系实时荧光PCR和数字PCR特异性检测方法符合转基因检测的要求。     

转基因大豆MON89788实时荧光PCR检测方法的建立

为实现转基因大豆MON89788的标识管理,针对转基因大豆MON89788的品系特异性序列设计引物和TaqMan探针,建立转基因大豆MON89788实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法,并对该方法的特异性、灵敏度和重复性进行检测。结果显示：建立的转基因大豆MON89788实时荧光PCR检测方法能扩增出127 bp的产物,特异性强,灵敏度达到0.1%,约为40 个单倍体基因组拷贝,检测重复性好,可成功应用于实际样品检测。因此,建立的转基因大豆MON89788实时荧光PCR检测方法可以应用于转基因大豆MON89788大豆及其制品的检测。     

应用多重PCR技术快速筛查食品中的转基因成分

以转基因植物中常见的3种筛选调控元件、2种标记基因和1种目的基因为检测靶标,建立了能在一次PCR扩增中同时检测6种转基因成分的多重PCR检测方法。结果表明,当多重PCR体系中Ca MV35S启动子、Fa MV35S启动子、NOS终止子、bar基因、NPTII基因、CP4-EPSPS基因的检测引物浓度分别为0.2、0.2、0.3、0.3、0.2、0.2μmol/L,退火温度为60℃时扩增效果最佳,利用优化后的六重PCR方法可从转基因玉米、大豆、棉花、油菜等各类样品中精准检测出相应的转基因成分,方法的检测灵敏度可达到0.1%。该方法为食品中常见转基因成分的筛查提供了一种高效的手段,在转基因产品的检测工作中具有较高的应用价值。     

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.     

Detection of genetically modified soya and maize: Impact of heat processing

The analysis of processed foods entails a number of complications, which negatively affect the performance of DNA based detection methods. Heat-processing methods viz. autoclaving and micro-waving, that mimic processing and manufacturing, as model unit operation systems were used to study their effect on the detection of genetically modified organisms (GMOs). This study confirms the premise that high temperature and/or pressure significantly reduce the level of detectable DNA. PCR methods were developed and adapted to target varying amplicon sizes of the trait, construct and event specific gene sequences that occur in MON-810 maize and Roundup Ready^® soybean. Integrity of DNA, recovery and PCR amplicon size (<200 bp) are major factors that direct the successful detection of GMOs in processed foods. The model systems used provide a platform to devise better strategies in developing detection protocols, especially for processed foods containing GMOs.     

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     

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.     

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     

Qualitative and Quantitative Detection of Protein and Genetic Traits in Genetically Modified Food

Due to the market introduction of genetically modified organisms (GMOs) in crops, foods, and ingredients, legislation worldwide came face to face with the question of the use and labeling requirements on GMO crops and their derivatives. In this review, protein- and DNA-based methods, such as enzyme-linked immunosorbent assay, western blots, and qualitative and quantitative polymerase chain reaction PCR (Q-PCR) are reviewed. Qualitative detection methods for genetically modified (GM) sequences in foods have evolved rapidly during the past years. The sensitivity of these systems is extremely high, even for processed foodstuffs. However, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. The recently introduced labeling threshold for GMOs in food ingredients by the European Union has forced official food control laboratories to apply quantitative PCR methods. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are discussed. As quantitative GMO detection methods measure GMO contents of samples in relation to reference material, priority must be given to international agreements and standardization on certified reference materials. The rapidly increasing number of GM foods on the market demands the development of more advanced multidetection systems, such as microarray technology. Challenges and problems arising from the inability to detect GM foods for which the modified sequence is unknown, the lengthy standardization procedures, and the need to continuously update databases comprising commercially available GM foods and the respective detection strategies are also discussed.     

Statistical Evaluation of Real-Time PCR Protocols Applied to Quantify Genetically Modified Maize

Real-time PCR (RTi-PCR) is the technique of choice for event-specific quantification of genetically modified organisms (GMOs) by determining the amount of event with respect to a species-specific reference gene. Reference genes can be amplified from the genome extracted from Certified Reference Materials (CRMs) or from ad hoc designed plasmids. In the present study, we statistically evaluate the performance of RTi-PCR protocols for GM maize MON810 event by using both genomic DNA from conventional CRMs and a plasmid containing sequences representative of four maize species-specific reference genes. The significance of simple and interaction effects of several variables included in the experimental design on DNA quantification methods and RTi-PCR were evaluated and discussed. Statistically significant differences on Ct values may have an impact on the GMOs quantification and consequently on the compliance of GM quantification-established legal thresholds. Our results confirm the reliability of the plasmid as alternative calibrant for the calculation of GMOs copy number.     

Comparison of High-throughput and Manual DNA Extraction Methods for the Qualitative and Quantitative Analysis of MON810 Maize

PCR-based methods are widely used in the European Union and in other countries for the detection, identification, and quantification of genetically modified organisms (GMOs). The preparation of good-quality DNA from plant samples for GMO detection can be a challenging task, particularly if the DNA will be used for quantitative analysis. Two DNA extraction methods, namely manual (NucleoSpin Food kit from Machery-Nagel) and high-throughput partially automated (NucleoMag Plant kit from Machery-Nagel) methods, which utilize different DNA separation principles, were used for the isolation of DNA from maize flour samples. Despite the higher DNA recovery obtained using the high-throughput isolation method, a lower PCR efficiency was achieved, most likely due to the presence of PCR inhibitors in the extracts. We found both DNA extraction methods suitable for GMO analysis.     

Determination of eight genetically modified maize events by quantitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. This has necessitated the development of methods for detection and quantification of such materials. Here we present a robust, quantitative, 9-plex PCR method for event-specific detection of maize TC1507, MON863, MON810, T25, NK603, GA21, construct specific detection of BT11, BT176 and detection of the endogenous hmga maize reference gene. The method is suitable for quantification in the 0–2% range with a detection limit of approximately 0.1%. PCR is carried out in two stages. In the first stage, bipartite primers containing a universal 5′-sequence and a GMO specific 3′-sequence are used. In the second PCR stage only a universal primer is used. Trypsin digestion between the first and second PCR stages enhances signal strength and reproducibility. Probes hybridising to the PCR amplicons are then labelled by primer extension and detected by fluorescence capillary electrophoresis. Good agreement was observed in 76 of 80 determinations when 10 food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. The presented method is therefore suitable for quantification purposes for food and feed containing the most common maize GMOs.