转基因棉花MON88913转化体特异性定性、定量PCR检测方法

本文以我国批准商业化的转基因耐草甘膦棉花MON88913为研究对象,建立并验证了其转化体特异性定性、定量PCR检测方法.建立的定性PCR方法的检测极限是20个拷贝棉花单倍体基因组DNA,定量PCR方法的检测和定量极限分别是10和20个拷贝棉花单倍体基因组DNA.同时,我们组织了实验室5位研究人员对建立的定量PCR检测方法进行了协同验证.对5个盲样的定量分析结果显示与真实值的偏差介于1.59% 和10.12%之间,完全满足国际标准25%偏差范围的要求,完全可用于转基因棉花MON88913的实际样品检测.     

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     

Characterization and event‐specific quantitative detection of DAS‐59122‐7 maize insert with the application of plasmidic reference material

BACKGROUND: With the development of genetically modified organisms (GMOs), event‐specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed standard. RESULTS: The flanking regions of DAS‐59122‐7 maize were characterized by inverse PCR (I‐PCR). In the qualitative PCR assay, a duplex PCR was established with the event‐specific and taxon‐specific primers, and the limit of detection (LOD) was 1 g kg^?1 (approximates to 38 haploid genome copies). In the quantitative TaqMan® real‐time PCR assay, a plasmidic reference material was constructed by recombinant PCR and standard curves were set up. By using the plasmidic reference material, we obtained standard curves with good linearity and relatively high efficiency. The results indicated the usability of the plasmid as standard material. CONCLUSION: From above results, we believe that the developed event‐specific qualitative and quantitative PCR systems for DAS‐59122‐7 maize in this study are acceptable and suitable for DAS‐59122‐7 maize detection. Copyright © 2008 Society of Chemical Industry     

Event‐specific analytical methods for biotech maize MIR 604 and DAS‐59122‐7

BACKGROUND: It is very important to develop analytical methods for genetically modified organism (GMO) labelling systems and living modified organism (LMO) management. The polymerase chain reaction (PCR) is the most efficient DNA‐based analytical method for identifying and quantifying biotech crops. Qualitative PCR methods have been developed to detect the presence of biotech crops, while quantitative PCR methods have been developed to analyse the content of biotech crops. Analytical methods are now required for new biotech maize events, MIR 604 and DAS‐59122‐7. RESULTS: The event‐specific primers and probes were developed for qualitative and quantitative analysis of biotech maize MIR 604 and DAS‐59122‐7 based on the 3′ flanking regions. As a reference molecule, single standard plasmid was developed. The specificity of the qualitative primers was confirmed by the single PCR product, with limits of detection (sensitivity) of 0.01 and 0.05% respectively. In‐house validation of the quantitative methods was performed using six levels of mixing samples (0.1–10% w/w). As a result, the biases from the true value and the relative standard deviations were within the range of ± 30%. The limits of quantitation of the quantitative methods were all 0.1% for real time PCRs of MIR 604 and DAS‐59122‐7. CONCLUSION: In this study, event‐specific analytical methods were developed and applied to the qualitative and quantitative analysis of biotech maize MIR 604 and DAS‐59122‐7. Copyright © 2009 Society of Chemical Industry     

A cotton‐specific gene,stearoyl‐ACP desaturase,used as a reference for qualitative and real‐time quantitative polymerase chain reaction detection of genetically modified organisms

Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)‐based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl‐ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real‐time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties. Copyright © 2006 Society of Chemical Industry     

Monitoring of genetically modified food and feed in the Tunisian market using qualitative and quantitative real-time PCR

Genetically modified organisms (GMO) invade more and more the agricultural production in the world. Although there are no legislations on GM labeling and cultivation of GM crops in Tunisia, the present study aims to check the status of GMO in Tunisian market using qualitative and quantitative real time-PCR (QRT-PCR). Three-hundred-sixty five samples were collected and different DNA extraction methods were adapted and optimized. Specific primers targeting 35S promoter from Cauliflower mosaic virus (CaMV) and nopaline synthase terminator from Agrobacterium tumefaciens (At) were used for the detection of the GMO insert and Taxon specific primers for the detection of plant species. Validated Taqman® probes (EU-RL) targeting event specific regions of the maize events MON810, Bt11, and the soybean event RRS were used for the quantification studies. Seven food and feed products showed different amounts of RRS (1.9%), MON810 (2.1%), and Bt11 (1.6%). The results demonstrate for the first time the presence of GMO in Tunisian markets reinforcing the need for the development of accurate quantitative methods in routine analyses.     

Event-Specific Qualitative and Quantitative Detection in Transgenic Soybean OsDREB3 Based on the 5′ Flanking Sequence

With the development of genetically modified organisms, labeling regulations have been introduced that require appropriate detection methods. Event-specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed state-of-the art. Using adaptor PCR, we analyzed the flanking sequences of exogenous integrant in transgenic soybean OsDREB3, which has resistance genes. In this study 5′ region flanking sequences of exogenous gene were identified in the soybean OsDREB3 genome, which was integrated in chromosome 1 with an additional 394 bp insertion between soybean genomic DNA and exogenous gene. Based on these inserts and flanking sequences, the event-specific qualitative and quantitative PCR system was established for this line. In the conventional qualitative PCR assay, the event-specific primers designed were confirmed to be specific and the limit of detection (LOD) was 0.1%. In the quantitative real-time PCR assay, the LOD and the limit of quantity were 10 and 100 haploid genome copies, respectively. The goodness of the linearity and high efficiency of the PCR reaction indicated the utility of the established PCR system. This study provides two reliable methods and information for detection, identification, and quantification of the presence of non-authorized transgenic soybean OsDREB3.     

Event-specific detection of genetically modified wheat B73-6-1 based on the 3′-flanking sequence

In this study, 3′-flanking sequence between the host plant DNA and the integrated gene construct of pHMW1Dx5 vector in transgenic wheat B73-6-1 was revealed by means of adaptor PCR; thus, the fragment with the length of 3.1?kb was obtained, including a 190-bp wheat genomic DNA, which demonstrates that this HMW-GS gene was located on the wheat chromosome 3B. And the event-specific PCR primers were designed based upon the revealed 3′-flanking sequence; the conventional qualitative PCR and quantitative SYBR real-time PCR detection methods employing these primers were successfully developed. In conventional qualitative PCR assay, the limit of detection was 0.1?% for B73-6-1 wheat genomic DNA for one reaction. In the quantitative SYBR real-time PCR assay, the limit of detection and limit of quantification were 10 and 100 haploid genome copies, respectively. In addition, three mixed blind wheat samples with known B73-6-1 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event-specific real-time PCR detection systems were reliable, sensitive and accurate.     

The construction of pMD18-HT-Soybean as a calibrator plasmid and nested PCR assay for herbicide-tolerant soybeans

A multiple-target plasmid designated as pMD18-HT-Soybean, comprising part of a junction region of genetically modified soybean events A2704-12, A5547-127, MON89788 and GTS-40-3-2, and the endogenous soybean-specific lectin gene were constructed. The limit of detection for quantification of these four event-specific genes using pMD18-HT-Soybean plasmid was 20 copies. Furthermore, a nested PCR detection method was developed for the above four genetically modified soybean events. LOD value of nested PCR detection was 0.005 %. The above results demonstrated that the plasmid pMD18-HT-Soybean DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2, MON89788, A2704-12 and A5547-127 in food and feed products. And the validated results also indicated that the developed nested PCR method can be used for identification and quantification of four genetically modified soybean events and its derivates.     

转基因植物Real-time PCR方法检出限和定量限的研究

目前Real-timePCR(Rt-PCR)方法在转基因植物定量检测中应用最为广泛。有关该方法的检出限和定量限的计算并没有达成统一。本文采用统计模型对三种转基因植物进行实时荧光定量PCR方法检出限和定量限的研究。实验结果表明:转基因玉米NK603检出限5拷贝,定量限14拷贝;转基因棉花Mon15985检出限5拷贝,定量限12拷贝;转基因油菜Oxy235检出限4拷贝,定量限9拷贝。是利用统计学方法对转基因植物荧光定量PCR方法检出限和定量限研究的一个积极尝试,可用于其他转基因植物检测中检出限和定量限的确定。     

Establishment and evaluation of event-specific qualitative and quantitative PCR method for genetically modified soybean DP-356043-5

With the increasing development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. The polymerase chain reaction (PCR) technique has been the mainstay for GMO detection, especially for event-specific qualitative and quantitative PCR detection methods, which have become the internationally agreed state-of-art. This paper describes the character and event-specific quantitative detection method of DP-356043-5 (356043) soybean. In this research, the flanking regions were characterized by inverse PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right and left flanking sequences. In the qualitative PCR assay, PCR systems were established with the species-specific and event-specific primers, respectively. And event-specific primers were established on both right and left flanking sequences; the limit of detection (LOD) was both 0.05% (approximates to 42 haploid genome copies). In the quantitative TaqMan real-time PCR assay, we obtained standard curves with good linearity and relatively high efficiency of PCR. All the results indicated that the established event-specific qualitative and quantitative PCR systems for 356043 soybean in this study were reliable and suitable for 356043 soybean detection in mixed samples. Besides, based on the flanking sequence information we obtained, not only the qualitative and quantitative PCR system for detecting 356043 soybean can be established, but also some other novel event-specific detection methods using gene microarray, biosensor, etc., with target sequence on them can also be developed, which have a good value for detecting 356043 soybean.     

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。     

Simple,sensitive, accurate multiplex quantitative competitive PCR with capillary electrophoresis detection for the determination of genetically modified maize

Legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. To this end, we have developed a simple and accurate capillary electrophoresis multiplex quantitative competitive PCR (ce-mqcPCR) method for event-specific quantification of the five novel GM maize events DAS59122, LY038, MON88017, MIR604 and Event 3272. The method combines the simplicity of constructing multiple competitors in silico with the high resolution and sensitivity of fluorescence capillary electrophoresis and the use of an internal template reference amplicon. The competitors are synthesised commercially and added in equal amounts as a restriction enzyme-digested plasmid insert to the multiplex PCR. Quantification is performed by analysing the relative amounts of GMO and GMO competitor fragment pairs after capillary electrophoresis and correcting for differences in maize DNA by comparing with the internal reference gene amplicon. Since the competitors employ the same primers as their corresponding targets, all existing qualitative multiplex PCRs can in principle easily be converted to quantitative assays without changing primer sets or amplification conditions. The ce-mqcPCR method correctly determined 120 GMO templates in known mixed samples. No false-positive or false-negative signals were obtained.     

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     

A statistical approach for evaluation of PCR results to improve the practical limit of quantification (LOQ) of GMO analyses (SIMQUANT)

The predominant approach for quantification of genetically modified organisms (GMO) is the application of quantitative real-time PCR. However, for a large number of processed food and feed products, this approach is unsuitable, because they contain low amounts (mass) of amplifiable DNA. Here we present a novel approach, “Single molecule quantification” (SIMQUANT) for GMO quantification of samples with extremely low amounts of DNA. The approach is based on statistics and application of multiple qualitative parallel PCRs. Here the qualitative PCRs were done using real-time PCR setup, but this is not a requirement. The difference is that the quantitative real-time PCR requires that the target copy number exceeds the absolute limit of quantification (LOQ_abs) and provides quantity estimates by extrapolation from a linear regressional relationship between an observed cycle threshold (Ct) value and copy numbers, while with SIMQUANT the template DNA typically contains very few, e.g., one target copy per PCR volume and the quantity is estimated on the basis of observed ratio between positive and negative individual PCRs. The components of this analysis are the numbers of test samples, the size of each sample and the outcome in number and relative ratio of positive and negative test results. The approach results in a statistical estimate of the relative GM concentration based on the probability that one or more amplifiable GM template copies are present in discrete volumes. Thus, the approach is based on the ratio of discrete volumes without or with one or more PCR-amplifiable GM target copies. The approach described here can be used reliably with more than a 100-fold improvement of the practical LOQ (LOQ_pract) compared to real-time quantitative PCR based on standard curves.     

应用多重PCR-液相悬浮芯片技术检测转基因水稻品系

开展转基因水稻的多重聚合酶链式反应（polymerase chain reaction,PCR）液相芯片检测方法研究。针对科丰6号（KF6）、科丰8号（KF8）、华恢1号（BT63）、克螟稻（KMD1）、M12、T1C-19、T2A-1、LL62和LL601九种转基因水稻的侧翼序列,设计合成了在生物素标记的多重PCR扩增引物与固定在荧光编码微球上的探针,建立了2 个多重PCR-液相芯片检测体系,可同时检测出这9 种转基因水稻成分。结果表明,9 种转基因水稻的引物和探针都具有较高的特异性,各组引物/探针之间无交叉扩增和非特异杂交,且在多重PCR-液相芯片检测中9 种转基因水稻品系的相对检测灵敏度达到0.1%水平,符合欧盟和其他国家有关转基因产品标识的要求。本方法的检测效率和准确性均高于传统方法,可作为进出口转基因产品和国内转基因检测的有效方法。     

转基因大米TT51-1品系精准定量检测方法的比较研究

基于大米蔗糖磷酸合成酶(SPS)基因和TT51-1品系特异性基因序列筛选适用于数字PCR的内、外源基因特异性引物探针并建立转基因大米TT51-1品系的双重数字PCR定量方法。其定量的绝对灵敏度和相对灵敏度分别达2 copies/μL和0.1%。当样品中TT51-1转基因大米成分含量低至0.1%时,6次定量值的相对标准偏差在7.30%~18.63%之间,偏差在-8.77%~9.62%之间,精密度和稳定性均较为理想,而同样的引物探针所建立的实时荧光PCR方法定量的相对灵敏度仅达到1%。为促进该方法的标准化应用,将微滴式数字PCR平台上建立的定量方法在芯片式数字PCR平台上进行室内验证,结果表明该方法的定量精密度和准确性符合要求。该方法可应用于大米、稻谷及其初加工产品中TT51-1转基因大米成分的精准定量检测。     

A construct-specific qualitative and quantitative PCR detection method of transgenic maize BVLA430101

Transgenic phytase maize (Zea mays L.) line BVLA430101 was the first transgenic maize obtained the security certification in 2009 in China. However, the construct of the phytase gene expression cassette and the specific detection method have not been reported yet. In this study, the phytase gene expression cassette was identified, which include maize legumin promoter, signal peptide, phytase gene, and maize legumin terminator. The construct-specific qualitative and quantitative PCR methods of BVLA430101 maize were established based on the transition of signal peptide and phytase gene using a maize taxon-specific gene zSSIIb as the endogenous gene. The detection limit for the conventional qualitative PCR was 200 haploid genome copies of BVLA430101. The absolute limit of quantification of the real-time PCR was about 20 haploid genome copies. In addition, two known BVLA430101 contents (5 and 1%) of mixed genomic DNA (V/V) were quantified using the developed real-time PCR detection system, which indicated that the developed quantitative method can be employed reliably for transgenic phytase maize BVLA430101 measurement.     

LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62

Information on the insertion site and characterization of the transgene(s) in genetically modified organisms (GMO) is very important for safety assessment and identification of a GMO. The generation of such information in general and in particular in emergencies or rapid alert situations involving GMO greatly benefit from the availability of simple, efficient, and rapid approaches. Here, we report on the improvement of a restriction independent method named “Rapid Amplification of genomic DNA Ends” (RADE). The method was developed using maize event MON810 genomic DNA as a model system, testing a standard Taq polymerase or a blend of polymerases (standard Taq and proofreading Tgo polymerases (LT-RADE)). Both methods produce an initial single strand DNA, followed by nested PCR steps and yield easy-to-isolate DNA fragments for further manipulation. We showed that the application of the Taq/Tgo polymerase blend significantly increased the size of the obtained PCR products. Using LT-RADE, we could successfully isolate the flanking regions of the transgenic insert of the GM maize event MON810 and confirmed the existing data on the adjacent regions of the insert. In addition, application of our approach allowed to efficiently isolate and identify, for the first time, the DNA sequences surrounding the insert of GM rice event LLRICE62.     

Selection of a Taxon-Specific Reference Gene for Qualitative and Quantitative PCR Detection of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis>

Safflower (Carthamus tinctorius) is an emerging model plant for the transgenic modification of fatty acid composition and the production of pharmaceuticals, proteins, or enzymes. Safflower is also a traditional Chinese medicine and is often used as a fake saffron product. Reliable detection of an endogenous reference gene is indispensable for the supervision of genetically modified safflower. Such an endogenous reference gene can also be used to specifically identify safflower ingredient in complex mixtures such as medicine or food. In this study, we identified and validated the CTOS gene as an endogenous reference for safflower. Conventional and real-time polymerase chain reaction (PCR) methods for detecting the CTOS gene sequence showed high interspecies specificity and intra-species stability. The lowest copy number detectable by conventional PCR was 10 haploid copies. The limit of detection and limit of quantification for the real-time PCR assay were estimated to be five and 40 haploid genome copies, respectively. Standard curves established for the real-time PCR assay exhibited good linearity (R ² > 0.99) between the cycle threshold (Ct) values and the initial template copies. The developed conventional and real-time PCR assays were validated in routine analysis of the safflower ingredient in commercial Chinese medicines. In conclusion, the developed quantitative PCR methods were sufficiently specific and sensitive to be used in safflower genomic DNA quantification and safflower ingredient identification.