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1.
抗虫玉米MON89034转化体特异性PCR检测技术研究 总被引：5，自引：2，他引：3
根据抗虫玉米MON89034外源插入片段5’端与植物基因组连接区序列设计特异性引物,并进行PCR扩增,预期产物大小为455 bp。以zSSIIb基因作为内标准基因,建立了转基因玉米MON89034转化体特异性定性PCR检测方法。对该方法进行重现性、特异性和灵敏度测试,结果表明:该方法能够特异性检测出MON89034转化体,以100 ngDNA为模板,该方法的检测灵敏度达到0.1%,约为40个起始模板拷贝。复合PCR检测结果还表明,在同一PCR反应管中可实现对zSSIIb基因和MON89034的同时检测。 相似文献
2.
应用多重荧光PCR快速筛查作物中转基因成分研究 总被引：1，自引：0，他引：1
通过对我国批准进口的和获得农业转基因生物安全证书的转基因玉米转化体的序列分析发现,除DAS40278-9和BLVA430101外,CaMV35s启动子、NOS终止子、Cry1Ab/Ac基因和pat基因覆盖了21种玉米转基因转化体。通过引物组合筛选、反应体系优化、灵敏度测试、适用性测试等实验,建立了基于4个通用筛选元件及zSSIIb内源基因的五重荧光PCR和基于转化体特异性序列的二重荧光PCR检测转基因成分方法体系。方法参数测定结果表明,此方法特异性强、稳定性好,检测灵敏度达到0.05%。同时,此方法体系不仅适用于转基因玉米成分筛查,对大豆、水稻等多种作物进行转基因成分筛选鉴定也有良好的适用性。 相似文献
3.
瑞丰125为农业农村部首批获得生产应用农业转基因生物安全证书的抗虫耐除草剂转基因玉米之一。为给监管转基因生物安全提供支撑，利用重组酶聚合酶扩增（recombinase polymerase amplification, RPA）技术，针对瑞丰125的转化体插入位点序列设计了引物并进行筛选，对反应的温度、时间和引物浓度进行了优化。结果显示，RPA体系在30～45℃范围内，20 min即可有效扩增，比PCR检测时间大大缩短。引物浓度会影响RPA的扩增效率，引物终浓度为0.35μmol/L时扩增效果最好。同时对RPA检测方法的特异性、灵敏度等进行考察，结果表明该检测方法的特异性良好，检测灵敏度可达到36拷贝。该恒温快速的检测方法为瑞丰125的快速检测提供依据，为商业化转基因作物的监管提供支撑，有望用于转基因成分的现场快速检测。 相似文献
4.
转基因玉米MON810(Yield Gard R)是孟山都公司通过DNA重组技术和微注射轰击研发的一种具有对欧洲玉米螟(ECB;Ostrinia nublialis)有特殊抗性的转基因玉米品系,目前在世界各地已经得到广泛种植,为加强对该品系玉米的安全管理,本研究旨在建立MON810品系玉米的转化事件特异性定性PCR检测方法。根据MON810的插入序列信息,在3′端的侧翼序列处设计定性PCR检测的引物,检测MON810在其他几种常见转基因作物混合样品的特异性。结果表明,该方法具有很好的特异性。同时检测该引物系统的扩增灵敏度,结果表明,检测引物的灵敏度可达0.1%。建立的MON810特异定性PCR检测方法经全国7家实验室的验证,进一步证实该方法能够特异地检测出样品中的MON810转化事件,检测方法的灵敏度可达0.1%,且检测结果具有良好的可重复性和可重现性。MON810转化事件定性PCR检测方法的建立可满足于抗虫转基因玉米MON810及其衍生品种生物安全管理的需要。 相似文献
5.
环状等温扩增技术快速检测转基因玉米MON863的研究 总被引：3，自引：1，他引：2
采用环状等温扩增技术(Loop-Mediated Isothermal Amplification,LAMP),针对转基因玉米品系MON863中玉米基因组与外源基因结合处设计4条特异性引物,建立MON863的快速检测技术体系,并对时间、温度等反应条件及体系灵敏度、特异性等进行探索。结果表明:该检测体系最适反应温度为63℃,反应时间60 min,灵敏度是常规定性PCR的10倍。该检测方法具有高度的特异性和稳定性,操作方便、简单、省时。 相似文献
6.
利用多重PCR技术快速检测五个转基因大豆品系 总被引：2，自引：0，他引：2
转基因大豆305423、MON89788、CV127、GTS40-3-2、356043作为商品化应用最为广泛的大豆品系,种植面积占世界转基因大豆总种植面积的80%以上。根据大豆内标准基因Lectin和5种转基因大豆品系的边界序列设计特异性引物。通过验证引物的适用性、特异性和灵敏度,优化多重PCR检测体系中不同引物的用量及反应退火温度,建立了能同时扩增大豆内源基因Lectin和5个转基因大豆品系的六重PCR检测体系。结果表明:确定的大豆内源基因和5个转基因大豆品系的引物具有很好的特异性,引物之间无交叉扩增和非特异性扩增,多重PCR方法的检测灵敏度达到0.1%。该方法可作为转基因大豆及其产品成分检测的辅助手段,快速检测转基因大豆及其产品中的相应品系。 相似文献
7.
应用单管巢式和半巢式PCR检测转基因玉米MON89034 总被引：1，自引：1，他引：0
根据MON89034玉米的5’端和3’端边界序列分别设计1组转化体特异性的巢式PCR引物,采用中途进退式PCR策略建立MON89034玉米的转化体特异性检测方法,扩增产物分别为491 bp和188 bp。以转基因玉米MON89034及8种其他转基因作物为材料,证明此方法对MON89034玉米具有高度特异性。灵敏度测试结果表明,此方法的相对检出限达到0.01%,绝对检出限为4个单倍体基因组拷贝数,比普通PCR提高了5倍。建立的单管巢式和半巢式PCR方法可准确、高效地检测转基因玉米MON89034及其产品。 相似文献
8.
转基因玉米育种研究进展 总被引：1，自引：1，他引：0
转基因性状包括抗虫、耐除草剂、耐干旱、雄性不育、高赖氨酸含量和耐热淀粉酶等。介绍了转基因玉米的转化途径、转基因玉米的功能基因及性状、转基因玉米的基因聚合方法以及目前美国市场上的主要转基因玉米性状、转基因玉米及其产品检测技术和转Bt基因玉米害虫的抗性治理,讨论转基因玉米基因专利失效以后的管理问题和转基因玉米基因发展趋势。 相似文献
9.
耐除草剂基因g10evo-epsps 在我国转基因农作物研发中常常被用作目标性状基因或筛选标记基因，因此
可作为转基因成分检测的重要筛查基因，但目前缺少相应的检测方法。本研究旨在建立g10evo-epsps 基因特异性
的实时荧光定量PCR（qPCR）检测方法，从而为转基因大豆的监测提供一种稳定可靠的方法体系。本研究基于转基
因大豆中的g10evo-epsps 基因序列设计了qPCR检测引物和探针，并对检测方法的特异性、灵敏度、准确度和精确性
进行了实验室内验证。结果显示，建立的g10evo-epspsqPCR检测方法能够特异性检测转基因大豆ZUTS-33中的
g10evo-epsps 目的基因；标准曲线分析表明，3次重复试验的扩增效率在90%以上，R2 均大于0.99；方法的检测限
（LOD）为不高于8拷贝，定量限（LOQ）推测为16拷贝；对含量为4.5%、2%、0.5%、0.09% 和0.045% 的转基因大豆
ZUTS-33基因组DNA进行准确度分析，发现该方法测定的平均值和预期值之间的偏差为0.00%~11.11%，3次重复
试验的RSDr为2.30%~17.10%。结果表明本研究建立的g10evo-epsps qPCR检测方法能够满足转基因大豆筛查检测
的要求，为转基因大豆监管和标识提供技术支撑。 相似文献
10.
为了防止国外商业化生产的转基因番木瓜流入国内市场,建立转基因番木瓜 55-1 转化事件定性 PCR 检测方法,对于保护国内消费者知情权意义重大。本研究以转基因抗环斑病毒番木瓜 55-1 为研究材料,利用外源基因和番木瓜基因组序列设计了 9 对特异性检测引物,通过特异性引物筛选、熔解曲线分析、退火温度优化、特异性验证、灵敏度分析及检测限验证,建立转基因番木瓜 55-1 转化事件定性 PCR 检测方法。结果表明：本研究筛选出的检测引物,可特异的检出转基因番木瓜 55-1 转化事件,引物的检测灵敏度达到了 0.1%的标准,高于欧盟0.9%的检测要求,完全可满足转基因检测标识制度的顺利实施。 相似文献
11.
ZHANG Xiujie JIN Wujun XU Wentao LI Xiaying SHANG Ying LI Sha OUYANG Hongsheng 《水稻科学》2019,26(4):248-256
Endogenous reference genes(ERGs) provide vital information regarding genetically modified organisms(GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified(GM)ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase(SPS),phospholipase D(PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs,a new ERG gene, phosphoenolpyruvate carboxylase(PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R~2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection,which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice. 相似文献
12.
《Journal of Cereal Science》2006,43(2):250-257
An event-specific detection method was developed based on the flanking sequence of an exogenous integrant in the transgenic maize MON863 which contains cry3Bb1 gene expressing a Bacillus thuringiensis Cry3Bb1 protein that is selectively toxic to a maize root worm pathogen. The 3′-integration junction between host plant DNA and integrated DNA of transgenic MON863 maize was isolated using thermal asymmetric interlaced (TAIL)-PCR. The event-specific primers and TaqMan probe were designed based upon the isolated 3′-integration junction sequence, and qualitative and quantitative PCR systems were established employing these designed primers and probe. In this system, the limit of detection of the qualitative PCR assay was estimated to be 40 initial haploid copies. The limit of quantitation of the quantitative PCR assay in authentic MON863 maize seeds was estimated to be approximately 80 haploid copies. GM MON863 contents were also quantified relative to endogenous maize starch synthase IIb (zSSIIb) gene DNA, and the results were expressed as the percentage of genetically modified MON863 maize DNA relative to the total content of maize DNA. All the results indicated that the established MON863 event-specific qualitative and quantitative PCR detection system based on the 3′-integration junction was reliable, sensitive and accurate. 相似文献
13.
转基因玉米特异性检测阳性标准分子的构建与应用 总被引：3，自引：1，他引：2
试验用PCR方法从玉米中扩增内源基因zSSIIb片段,并将其克隆到pMD18-T载体上,获得中间载体pMD-zSSIIb;根据Bt11和MON810玉米转化体特异性序列,分别设计带酶切位点的引物,扩增出Bt11和MON810转化体特异性产物;用相应的酶对pMD-zSSIIb和两种PCR产物进行酶切,分别将Bt11和MON810产物克隆到pMD-zSSIIb上,获得阳性标准分子pMD-ZB和pMD-ZM,并对其进行特异性测试。结果表明,获得的阳性标准分子可以作为转基因产品检测时的阳性对照。 相似文献
14.
Genetically modified crops are widely grown in the world today. Labeling is required when genetically modified organisms (GMOs) are placed on the market. There is a need to establish a specific method for the detection of genetically modified foods. MON863 transgenic maize containing a Cry3Bb1 sequence that produces insecticidal protein cry3Bb1 is a major GMO crop. In this paper, we report studies that designed specific PCR primers and TaqMan probes based upon the 5′-transgene integration sequence, and developed qualitative and quantitative PCR conditions using these primers and probes. We determined the 5′-transgene integration sequence using a ligation-mediated polymerase chain reaction (LM PCR) method. In qualitative PCR studies, the limit of detection (LOD) was 0.5% for MON863 in 100 ng genomic DNA. In the quantitative PCR assays, the limit of detection (LOD) and limit of quantitation (LOQ) are 10 and 100 haploid copies, respectively. Maize samples with different contents of genetically modified component were tested using the established TaqMan real-time PCR system. 相似文献
15.
耐草甘膦和耐草铵膦是转基因作物育种重要的目标性状。将耐草甘膦基因MC1-EPSPS构建到含有bar基因的植物表达载体pTF101.1中，通过农杆菌介导法转入玉米材料Hi-II中，从而获得兼具耐受草甘膦和草铵膦性状的转基因玉米材料 CM8401。目的基因PCR检测显示，MC1-EPSPS和bar基因稳定整合到玉米基因组中。目的蛋白试纸条检测结果显示，MC1-EPSPS蛋白和PAT蛋白在转基因玉米世代间中表达稳定。田间除草剂耐受性鉴定试验表明，转基因玉米CM8401对草甘膦和草铵膦都具有良好耐受性，可耐受4倍推荐中剂量的草甘膦和草铵膦。 相似文献
16.
《Journal of Cereal Science》2009,49(3):592-597
Genetically modified crops are widely grown in the world today. Labeling is required when genetically modified organisms (GMOs) are placed on the market. There is a need to establish a specific method for the detection of genetically modified foods. MON863 transgenic maize containing a Cry3Bb1 sequence that produces insecticidal protein cry3Bb1 is a major GMO crop. In this paper, we report studies that designed specific PCR primers and TaqMan probes based upon the 5′-transgene integration sequence, and developed qualitative and quantitative PCR conditions using these primers and probes. We determined the 5′-transgene integration sequence using a ligation-mediated polymerase chain reaction (LM PCR) method. In qualitative PCR studies, the limit of detection (LOD) was 0.5% for MON863 in 100 ng genomic DNA. In the quantitative PCR assays, the limit of detection (LOD) and limit of quantitation (LOQ) are 10 and 100 haploid copies, respectively. Maize samples with different contents of genetically modified component were tested using the established TaqMan real-time PCR system. 相似文献
17.
以若干定性PCR方法部颁标准对含有0.5％的4份不同转基因混合样品进行检测,先以通用元件标准中的CaMV35s启动子、NOS终止子对混合样品进行初步定性PCR筛选。结果表明,4份样品中都含有转基因成分。Bt基因特异性标准检测表明,3#和4#样品含有转基因抗虫水稻成分。构建特异性标准PCR检测表明,2#、3#和4#样品含有转基因GTS-40-3-2大豆成分。以MON810、Bt176、NK603转化体事件标准进行品系特异性PCR检测,结果证实：1#和4#样品中含有Bt176转基因玉米成分;3#样品中含有Mon810转基因玉米成分;4份样品中均不含NK603转基因玉米成分。说明农业部颁布的定性PCR方法标准能满足于对多种转基因混合样品的检测,且检测结果准确,可靠。 相似文献