转基因玉米LY038转化事件的特异性检测

转基因高赖氨酸玉米LY038在畜禽饲料中已得到广泛应用,但目前还没有关于其侧翼序列扩增及转化事件特异性定性PCR检测方法方面的报道.本研究采用修饰接头连接PCR(modified adapter-linked PCR,M-AL-PCR)技术获得了转基因玉米LY038的外源基因与玉米基因组之间的5′端侧翼序列.据此侧翼 序列设计其转化事件特异性引物,建立了转基因玉米LY038转化事件特异性定性检测方法,扩增片段175bp.以转基因玉米(Zea mays L.)LY038、MIR604、Bt176、Bt11、MON810、MON863、GA21、NK603、非转基因玉米、转基因水稻(Oryza sativa L.)Cry1C*、Cry2A*、转基因大豆(Glycine max)Roundup Ready和转基因油菜(Brassica campestris L.)GT73为材料,验证该方法具有高特异性及灵敏度,最低检测限约为0.1%.研究结果提示,该定性检测体系可准确、快速、高效的检测转基因玉米LY038及其产品.     

转基因水稻Bt汕优63的整合结构和品系特异性定量PCR方法

为更好地监测转基因抗虫水稻(Oryza sativa L.)Bt汕优63品系的存在,满足转基因标识管理的要求,同时也为了解决阳性植物基因组DNA不容易获得的问题,本研究采用热不对称交互PCR(TAIL-PCR)、Genome Walking和长链PCR(LD-PCR)方法测定了基因枪转化的转cry1Ab/cry1Ac融合基因水稻品系Bt汕优63的外源插入DNA全序列,构建了含有Bt汕优63 3′旁侧和水稻内标基因gos9序列的标准质粒分子pMDBt63作为标准物质,建立了Bt汕优63实时荧光定量PCR方法.外源插入DNA全长9 818bp,位于水稻基因组10号染色体(AP008214)第5348630位,由8个来源于两个转化质粒的DNA片段组成,其中两个cry1Ab/cry1Ac表达框以正向串连的方式插入.gos9和3′旁侧两个定量标准曲线的相关系数(R2)分别为0.9997和0.9992,扩增效率(E)分别为98.05%和99.46%.每反应100 ng模板DNA的检测限(LOD)为10拷贝,定量限(LOQ)为100拷贝.此外,对已知5%和1%转基因含量的混合样品DNA进行定量检测,结果的平均值分别为4.98%和1.07%.结果表明标准质粒pMDBt63作为标准物质建立的定量方法可用于Bt汕优63的定量检测.     

转基因棉花GHB119品系特异性定量PCR检测方法的建立

为了保障和促进我国口岸进口转基因产品安全相关法律法规的顺利实施,针对我国农业部未颁发农业转基因生物安全证书的棉花品系GHB119,本研究利用TaqMan实时荧光PCR(Real-time PCR)技术,根据转基因棉花(Gossypium sp.)GHB119 3'端外源插入片段与棉花基因组DNA之间的邻接区序列设计引物和探针,并对引物、探针以及扩增体系进行了筛选和优化,建立了转基因棉花GHB119品系特异性实时荧光定量PCR检测方法。结果表明,建立的检测方法特异于转基因棉花GHB119成分检测,检测下限(limit of detection,LOD)为10拷贝的基因组DNA,定量下限(limit of quantification,LOQ)为25拷贝GHB119基因组DNA,对盲样的定量结果显示,测定值与设定值间的偏差(bias)、标准偏差(standard deviation,SD)、相对标准偏差(relative standard deviation,RSD)等均在可接受范围内。本研究建立的GHB119品系特异性实时荧光定量PCR检测方法特异性好,灵敏度高,能够快速、准确地对混合样品中转基因棉花GHB119成分进行定量检测分析。     

转基因棉花MON757转化体特异性PCR检测方法及应用

转基因棉花(Gossypium hirsutum)MON757转化体是孟山都公司研发的转cryIAc基因的抗虫棉,在美国、加拿大、澳大利亚等国被批准种植或允许用作食品饲料原料,但在我国尚未获得批准种植和应用.目前国内外尚没有转基因棉花MON757特异性的纯杂合检测和定量检测方法报道.为了检测我国棉花中MON757转化体的存在情况,本研究以转基因棉花MON757转化体的插入位点基因组序列和外源插入片段/基因组连接区序列为靶标,建立了特异性的MON757转化体纯杂合定性PCR检测方法和实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测方法.所建立的MON757转化体纯杂合定性PCR检测方法的检测引物由分别位于MON757外源基因插入位点两侧基因组和外源插入片段上的3条引物组成,能准确地鉴别棉花植株和种子中MON757转化体的纯合、杂合状态.建立的MON757转化体特异性的qRT-PCR检测方法具有良好的可重复性和灵敏度,其检测下限(limit of detection,LOD)为11个拷贝,定量下限(limit of quantitative,LOQ)为44个拷贝.用此方法对2014年湖北省的49份商品棉花种子进行了检测,结果有5份种子样品检测到MON757转化体.采用MON757转化体纯杂合定性PCR检测方法对这5份种子样品各60个单粒分别进行了检测,同时采用qRT-PCR检测方法对这5份种子样品进行定量测定.结果表明,有1份含量在1.5％左右,4份含量超过了20％,两种方法的测定结果基本一致.本研究建立的MON757转化体纯杂合定性PCR检测方法和qRT-PCR检测方法在田间棉花植株和种子中MON757转化体纯杂合状态的测定以及混合样品中MON757转化体含量的测定方面都具有重要的应用价值.     

一种检测转Bt基因抗虫棉新棉33B和GK-12的PCR方法

测定了转基因抗虫棉新棉33B和GK-12的Bt基因表达盒的序列,发现它们在Bt基因和Bt基因与终止子连接区的序列存在差异,而在启动子与Bt基因连接区的序列完全一致。基于这种结构上的差异,设计3条特异性引物MG-P1、MG-P2和MG-P3,建立了检测这两个转基因抗虫棉的双重PCR方法。采用建立的方法,检测了40个转基因棉花样品,其中,只含有新棉33B的Bt基因结构的样品数为32个;只含有GK-12的Bt基因结构的样品数为6个;同时含有两者结构的样品数为2个。     

实时荧光PCR检测转基因大豆外源基因的拷贝数

快捷、准确地检测转基因植物中外源基因的拷贝数,是转基因生物育种的重要研究内容,具有较大的应用前景。本研究以7个抗虫BT(Bacillus thuringiensis)和抗草甘膦EPSPS-G10转基因大豆事件为材料,以SYBR Green I为荧光染料,利用实时荧光定量PCR方法,根据PCR反应获得的Ct值与起始模板数的对数值存在线性反比关系这一原理,建立了相关性系数在0.99以上的模板定量标线。通过目的基因与内参基因——大豆肌动蛋白编码基因ACTIN的起始模板量比较,估算出各株系的目的基因拷贝数。数据显示,株系1、2、3和4的2个基因均为单拷贝,株系6的2个基因均有2个拷贝,株系5和7的2个基因的拷贝数为3。利用Southern印迹方法对材料1~4的拷贝数进行验证,结果表明,两者的检测结果基本一致,证明实时荧光PCR检测法是大豆外源基因拷贝数检测中快速有效的方法。实时荧光PCR高效快速检测基因拷贝数,对于大规模转基因株系的外源基因拷贝数检测应用具有重大意义。     

转基因产品检测技术研究进展

对转基因产品的定性PCR、定量PCR、酶联免疫吸附及其它检测技术进行了系统阐述,综述了转基因产品品系特异性检测技术、内标基因、标准物质及影响检测的抑制因子等已2v取得研究进展,指出了当前转基因产品检测技术存在的问题及未来的发展前景。     

转基因大豆GTS40-3-2转化事件特异性PCR检测

GTS40-3-2是抗草甘膦转基因大豆,为建立GTS40-3-2大豆转化体特异性PCR检测方法,本研究以GTS40-3-2标准品为实验材料,根据已公布转基因大豆GTS40-3-2基因与大豆基因组连接序列信息,利用Primer5.0软件设计了5对品系特异性引物,对每对引物进行了退火温度、特异性及扩增效率的PCR检测,结果显示,5对特异性引物均能够从GTS40-3-2中扩增出大小约279bp、238bp、470bp、490bp和257bp的预期产物,可用于特异性检测转基因大豆GTS40-3-2转化事件。以转基因大豆GTS40-3-2含量为5%、2%、1%、0.5%和0.1%的标准品进行PCR灵敏度检测,结果表明5对引物的检测灵敏度均能达到0.1%。通过荧光定量PCR对5对特异性引物的Ct值与溶解曲线比较,最后选择出RRS2引物对为转基因大豆GTS40-3-2品系特异性检测的最适引物。本文结果将为我国未来转基因生物产品成分检测提供科学合理的实验参考。     

利用微滴数字PCR分析转基因生物外源基因拷贝数

微滴数字PCR(droplet digital PCR,ddPCR)是一种基于泊松分布原理的核酸分子绝对定量技术,在核酸分子的绝对计数/定量领域具有极大的应用潜力。本研究基于ddPCR平台,以转基因水稻(Oryza sativa)T1c-19和转人乳铁蛋白基因基因山羊(Capra hircus)134为例,建立了转基因生物(genetically modified organisms,GMOs)外源基因拷贝数分析方法,并比较了其与传统的实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和Southern blot方法的准确性。实验数据表明,T1c-19的杀虫晶体蛋白基因(insecticidal crystal protein,Cry1C*)在qRT-PCR和ddPCR测定结果比较一致,约为2拷贝,但已报道的Southern blot分析结果为1拷贝;ddPCR对bar基因的分析结果高于qRT-PCR,分别为2.09拷贝和1.51拷贝。转人乳铁蛋白基因(human lactoferrin,HLF)山羊134在qRT-PCR和ddPCR的分析结果基本一致,均测得含有1拷贝的HLF基因。研究结果表明,微滴数字PCR方法是一种经济、快速和准确的外源基因拷贝数分析新方法,灵敏度和准确性高,将会在拷贝数分析中广泛应用。     

转基因玉米多重PCR-基因芯片联用检测方法的研究

根据七种转基因玉米的重组DNA结构分别对Bt11、Bt176 、Mon810 、Mon863 、TC1507、 GA21 和NK603设计转化体特异性引物,进行多重PCR检测。在此基础上分别设计和筛选了七种转基因玉米转化体特异性oligo探针,制备转基因玉米的寡核苷酸芯片。实验表明,该探针特异性好,同常用的凝胶电泳检测方法相比,芯片杂交的灵敏度（0.01%）,优于凝胶电泳检测（0.1%）,由于采用了多重PCR技术一次可同时检测多个基因,提高了检测的准确率和效率。     

Establishment and application of event-specific polymerase chain reaction methods for two genetically modified soybean events, A2704-12 and A5547-127

For implementation of the issued regulations and labeling policies for genetically modified organism (GMO) supervision, the polymerase chain reaction (PCR) method has been widely used due to its high specificity and sensitivity. In particular, use of the event-specific PCR method based on the flanking sequence of transgenes has become the primary trend. In this study, both qualitative and quantitative PCR methods were established on the basis of the 5' flanking sequence of transgenic soybean A2704-12 and the 3' flanking sequence of transgenic soybean A5547-127, respectively. In qualitative PCR assays, the limits of detection (LODs) were 10 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127. In quantitative real-time PCR assays, the LODs were 5 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127, and the limits of quantification (LOQs) were 10 copies for both. Low bias and acceptable SD and RSD values were also achieved in quantification of four blind samples using the developed real-time PCR assays. In addition, the developed PCR assays for the two transgenic soybean events were used for routine analysis of soybean samples imported to Shanghai in a 6 month period from October 2010 to March 2011. A total of 27 lots of soybean from the United States and Argentina were analyzed: 8 lots from the Unites States were found to have the GM soybean A2704-12 event, and the GM contents were <1.5% in all eight analyzed lots. On the contrary, no GM soybean A5547-127 content was found in any of the eight lots. These results demonstrated that the established event-specific qualitative and quantitative PCR methods could be used effectively in routine identification and quantification of GM soybeans A2704-12 and A5547-127 and their derived products.     

Event-specific qualitative and quantitative polymerase chain reaction analysis for genetically modified canola T45

Polymerase chain reaction (PCR) methods have been the main technical support for the detection of genetically modified organisms (GMOs). To date, GMO-specific PCR detection strategies have been developed basically at four different levels, such as screening-, gene-, construct-, and event-specific detection methods. Event-specific PCR detection method is the primary trend in GMO detection because of its high specificity based on the flanking sequence of exogenous integrant. GM canola, event T45, with tolerance to glufosinate ammonium is one of the commercial genetically modified (GM) canola events approved in China. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of T45 canola was cloned and revealed by means of TAIL-PCR. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence, and qualitative and quantitative TaqMan real-time PCR detection assays employing these primers and probe were developed. In qualitative PCR, the limit of detection (LOD) was 0.1% for T45 canola in 100 ng of genomic DNA. The quantitative PCR assay showed limits of detection and quantification (LOD and LOQ) of 5 and 50 haploid genome copies, respectively. In addition, three mixed canola samples with known GM contents were detected employing the developed real-time PCR assay, and expected results were obtained. These results indicated that the developed event-specific PCR methods can be used for identification and quantification of T45 canola and its derivates.     

Qualitative and quantitative event-specific PCR detection methods for oxy-235 canola based on the 3' integration flanking sequence

As more genetically modified plant events are approved for commercialization worldwide, the event-specific PCR method has become the key method for genetically modified organism (GMO) identification and quantification. This study reveals the 3' flanking sequence of the exogenous integration of Oxy-235 canola employing thermal asymmetric interlaced PCR (TAIL-PCR). On the basis of the revealed 3' flanking sequence, PCR primers and TaqMan probe were designed and qualitative and quantitative PCR assays were established for Oxy-235 canola. The specificity and limits of detection (LOD) and quantification (LOQ) of these two PCR assays were validated to as low as 0.1% for the relative LOD of qualitative PCR assay; the absolute LOD and LOQ were low to 10 and 20 copies of canola genomic DNA in quantitative PCR assay, respectively. Furthermore, ideal quantified results were obtained in the practical canola sample detection. All of the results indicate that the developed qualitative and quantitative PCR methods based on the revealed 3' integration flanking sequence are suitable for GM canola Oxy-235 identification and quantification.     

Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule

With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.     

Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1xRF1 and MS1xRF2

Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1xRF1 and MS1xRF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1xRF1 and MS1xRF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1xRF1 and MS1xRF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.     

Indicated detection of two unapproved transgenic rice lines contaminating vermicelli products

We analyzed the DNA fragments extracted from four rice vermicelli products. The Bacillus thuringiensis (Bt) rice line, which has a construct similar to the GM Shanyou 63 line, was detected in some vermicelli products by identification of the junction region sequence between rice Act1 promoter and the Cry1Ac gene, and that between Cry1Ac and nos. In addition, we also detected a different Bt rice line by means of the junction region sequence between the maize ubiquitin promoter and cry1Ab gene and that between the cauliflower mosaic virus 35S promoter and the hygromycin phosphotransferase in some vermicelli products. Accordingly, we for the first time have detected the two transgenic Bt rice lines contaminating rice vermicelli samples. Furthermore, we developed a duplex real-time polymerase chain reaction (PCR) method for the simultaneous detection of both Bt rice lines.     

cry1Ac/cpti转基因大米水稻土中微生物群落的多样性研究

Two types of cry1Ac/cpti transgenic rice(GM1 and GM2)and their parental non-cry1Ac/cpti rice(CK1 and CK2)were planted in the field at Wufeng,Fujian Province,China for four years to investigate the influence of genetically modified rice on diversity of bacterial and fungal community in the paddy soil.The community composition and abundance of bacteria or fungi in the paddy soil were assessed at different growth stages of rice by denaturing gradient gel electrophoresis and real-time polymerase chain reaction based on 16S rRNA gene or SSU rRNA gene in the 4th year after the experimental establishment.The composition of bacterial or fungal community changed during rice growth,while no significant differences were observed between the fields cultivated with GM1and CK1,or between the fields cultivated with GM2 and CK2 in either bacterial or fungal community composition.The copy numbers of bacterial 16S rRNA gene in the soils with CK1,CK2,GM1 and GM2 ranged from 5.64×1011to 6.89×1011copies g-1dry soil at rice growth stages,and those of fungal SSU rRNA gene from 5.24×108to 8.68×108copies g-1dry soil.There were no marked differences in the copies of bacterial 16S rRNA gene or fungal SSU rRNA gene between CK1 and GM1 or between CK2 and GM2at any growth stage of rice.Planting cry1Ac/cpti transgenic rice had no significant effect on composition and abundance of bacterial and fungal community in paddy soil during the rice growing season at least in the short term.     

Event-specific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray

With the increasing development of genetically modified organism (GMO) detection techniques, the polymerase chain reaction (PCR) technique has been the mainstay for GMO detection. An oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detect ten or more targets synchronously. In this research, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. A commercial GM soybean (GTS 40-3-2) and six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes.     

Method for the detection of synthetic cry3A in transgenic potatoes

All transgenic cultivars of potatoes registered in Canada and the United States have been modified to express a synthetic cry3A gene as a means of conferring resistance against the Colorado potato beetle, an important economic pest of potatoes. A PCR method was developed to amplify a 499 bp region of the synthetic cry3A gene. Using this method, synthetic cry3A could be detected in six different transgenic cultivars. Positive results could be confirmed with PvuII restriction digestion of the PCR-generated amplicon, which resulted in two fragments that were 283 and 216 bp in size. Of the 52 tuber extracts tested with this method, no false positive or false negative results were obtained, suggesting the method could be used with a high degree of accuracy. The absolute limit of detection was the number of cry3A copies present in one or perhaps two haploid copies of the potato genome. The practical limit of detection in tubers on a fresh weight basis was 0.02% for the NL 10-SUP and 0.01% for the remaining cultivars. Synthetic cry3A could also be detected in processed food products such as potato chips, shoestring potatoes, and frozen French fries. The method was suitable for screening potato tuber lots and some processed foods for the presence of synthetic cry3A.     

Applicability of plasmid calibrant pTC1507 in quantification of TC1507 maize: an interlaboratory study

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of DNA plasmids as calibrants is becoming essential for the practical quantification of GMOs. This study reports the construction of plasmid pTC1507 for a quantification assay of genetically modified (GM) maize TC1507 and the collaborative ring trial in international validation of its applicability as a plasmid calibrant. pTC1507 includes one event-specific sequence of TC1507 maize and one unique sequence of maize endogenous gene zSSIIb. A total of eight GMO detection laboratories worldwide were invited to join the validation process, and test results were returned from all eight participants. Statistical analysis of the returned results showed that real-time PCR assays using pTC1507 as calibrant in both GM event-specific and endogenous gene quantifications had high PCR efficiency (ranging from 0.80 to 1.15) and good linearity (ranging from 0.9921 to 0.9998). In a quantification assay of five blind samples, the bias between the test values and true values ranged from 2.6 to 24.9%. All results indicated that the developed pTC1507 plasmid is applicable for the quantitative analysis of TC1507 maize and can be used as a suitable substitute for dried powder certified reference materials (CRMs).