双重数字PCR在转基因大豆检测中的应用

利用微滴式数字PCR(droplet digital PCR, ddPCR)平台建立针对MON87705、MON87769、DP356043三种转基因大豆中外源基因的双重PCR检测方法。利用双重数字PCR方法检测特异性、定量范围等参数,优化所用引物探针组合及实验体系程序,检测外源基因与内标准基因的拷贝数。结果表明,所用引物探针组合在数字PCR方法中仅对目标大豆品系有荧光信号,具有特异性,可用于转基因大豆品系的筛选与鉴别。检测了大豆的转基因成分含量,结果与材料标准品参数基本一致,并根据结果设定定量检测限为0.5%,定性检测限为0.05%,可满足低纯度样品检测的需求。双重数字PCR体系能够准确且稳定的满足实际检测需要,在实际应用上具有良好的发展前景。     

转g10-epsps基因耐除草剂大豆ZUTS-33转化体特异性检测方法的建立

转g10-epsps基因耐除草剂大豆ZUTS-33是由浙江大学研发的耐除草剂大豆品系,目前已进入生产性试验阶段。到目前为止尚无文献报道对该转基因新品种的检测方法,因此亟需建立精准的定量检测方法为农业转基因生物安全管理提供技术支持。根据耐除草剂大豆ZUTS-33品系外源基因插入位点特异序列设计引物和TaqMan探针,利用优化的实时荧光定量PCR检测方法评价该引物对和探针的特异性、准确度、精确度和重复性,并确定此检测方法的检测极限（limit of detection,LOD）和定量极限（limit of quantity,LOQ）。实验结果显示,研究所建立的转基因大豆ZUTS-33转化体特异性实时荧光定量PCR检测方法具有高度的品系鉴定特异性,准确度、精确度均符合要求,重复性较好,且检测方法的LOD达到20拷贝,LOQ达到40拷贝。研究结果为转g10-epsps基因耐除草剂大豆ZUTS-33的身份识别和检测监测提供了有效的方法。     

转基因抗虫耐除草剂玉米MON87411精准定量检测方法的建立

【目的】抗虫耐除草剂玉米MON87411是孟山都远东有限公司利用农杆菌介导方法研发的玉米转化体,已获得我国进口用作加工原料的农业转基因生物安全证书。为满足生物安全监管的要求,亟需建立该转化体的定量检测方法。【方法】根据抗虫耐除草剂玉米MON87411的两端旁侧序列信息设计引物和Taqman探针,进行引物筛选、特异性检测、PCR体系优化、标准曲线建立、正确度及精密度检测、检出限及定量限测试、微滴数字PCR方法验证等。【结果】该方法能特异、定量地检测出抗虫耐除草剂玉米MON87411转化体成分,检出限低至10拷贝,定量限为40拷贝。对测试样品定值准确,经微滴数字PCR方法验证结果一致。【结论】本方法为该新品种转基因玉米品系的精准定量提供了一种新的检测方法,为生物安全监管提供了有效的技术支撑。     

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。     

基于RNAi技术的转基因玉米逆转录数字PCR检测方法

针对基于RNAi技术的转基因玉米品系,研究并建立了一套逆转录芯片式数字PCR(dPCR)定量检测该品系玉米双链RNA的方法。研究内容主要包括RNA提取方法、引物探针设计、逆转录方法、dPCR反应条件及体系等方面的探索和优化。该方法的绝对定量限为2.5 copies/μL,RSD为12.4%;检测低浓度实际样品达21.7 copies/μL时,相对偏差为2.7%,RSD为14.6%,满足国际上转基因定量结果RSD≤25%的要求。将该方法用于基于RNAi技术转基因作物的定量检测,将为我国相关转基因作物的安全评价提供了精确可靠的技术手段。     

转基因棉花MON88701品系特异性实时荧光PCR检测方法的建立

【目的】建立转基因棉花MON88701品系特异性实时荧光聚合酶链反应(polymerase chain reaction,PCR)检测方法。【方法】利用实时荧光PCR技术,根据转基因棉花MON88701品系特异性序列设计引物和探针,建立转基因棉花MON88701实时荧光PCR检测方法,并测定本方法的灵敏度、特异性及可重复性。【结果】建立的检测方法特异于转基因棉花MON88701成分的检测,灵敏度测试表明其定量下限为34拷贝;重复性试验显示其相对标准偏差在可接受范围内。【结论】本研究建立的转基因棉花MON88701品系特异性实时荧光PCR检测方法具有良好的特异性和高灵敏度,适合对转基因棉花MON88701品系进行检测。     

醛基片PCR芯片技术在转基因玉米品系检测中的应用研究

针对9种转基因玉米品系Bt11、TC1507、Bt176、MON810、MON863、GA21、NK603、Mon88017、MIR604进行醛基片PCR芯片高通量检测和条件优化。特异性和灵敏度试验表明,醛基片PCR芯片的特异性较好,能应用于转基因玉米品系鉴定。而该方法应用于转基因检测的灵敏度为5%,有待于后续试验进一步优化。     

用高效液相色谱测定重组人尿激酶原制剂的蛋白含量

目的:用RP-HPLC方法对注射用重组人尿激酶原制剂蛋白含量进行定量分析。方法:用反相C18柱、0.1%TFA水溶液与0.1%乙腈进行梯度洗脱,280nm波长紫外检测器监测;以重组人尿激酶原同质标准品作为对照品,根据进样量和相应的峰面积建立标准曲线方程,将待测定样品的峰面积代入标准曲线方程,可测得蛋白含量。结果:按照方法学验证要求对此方法进行了专属性、检测限、定量限、线形、精密度(重复性、中间精密度)、准确度(回收率)考察,线性范围为9～27μg,回收率在97%以上,RSD2.0%,完全满足对制剂蛋白的定量需求。结论:本方法准确,适用于注射用重组人尿激酶原成品制剂蛋白定量测定。     

转基因水稻“科丰6号”实时荧光PCR定性定量检测方法研究

旨在建立转基因水稻"科丰6号"外源基因和边界序列的实时荧光PCR检测方法,为科丰6号定性定量检测提供技术支持。根据外源基因和边界序列信息,设计实时荧光PCR探针引物,优化体系,对不同转基因产品和不同转基因含量的"科丰6号"水稻进行检测。结果显示,所设计的引物探针具有很好的特异性,与其他转基因水稻品系、转基因玉米、转基因棉花、转基因番茄和非转基因水稻均无非特异性反应,对转基因水稻"科丰6号"的检测灵敏度达到0.01%。建立的科丰6号实时荧光PCR检测方法重复性好、灵敏度高,能够达到目前国际上转基因产品定量检测的标准,为该水稻品系的定性定量检测提供技术支持。     

基于数字PCR的转基因水稻LL62品系精准定量检测方法建立

为了促进转基因产品标识制度的顺利实施,对未获我国农业部批准的转基因水稻LL62品系进行精准定量检测方法研究,以保障我国进出口转基因农产品的检测监管。基于转基因水稻LL62品系外源插入片段和水稻基因组DNA的3'端旁侧序列设计数字PCR检测体系,建立了精准定量检测方法。结果显示,设计的引物探针反应效率高,基于此建立的数字PCR方法高度特异于转基因水稻LL62品系检测;方法可重复性好,生成微滴数相对标准偏差(RSD)值介于0.60%-11.11%;准确度高,3个混合样品(10%、1%和0.1%)测定值与真实值之间的偏差(bias)分别为0.12、0.09和0.10,相对标准偏差(RSD)介于0.09%-10.31%。建立的转基因水稻LL62品系微滴数字PCR定量检测方法简便快捷、特异性强、重复性好、准确度高,可准确有效地对农产品和食品中转基因水稻LL62成分进行精准定量分析。     

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.     

实时荧光定量PCR方法快速检测转基因大豆

针对转基因大豆中普遍含有的35S启动子进行引物设计,以双链DNA染料SYBR GreenⅠ为荧光标记物,利用实时荧光定量PCR方法对大豆样品进行检测。该法检测转基因大豆的检测低限为0.005 nmol/L的35S启动子,线性范围达3个数量级,可快速区分转基因大豆和非转基因大豆,具有快速、简便、灵敏、安全、高通量、低成本等优点,可推广用于转基因植物产品的快速定量检测。     

Identification of genetically modified vegetable sources in food and feed using hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by the hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in feed and food products. The biochip contained 22 immobilized probes intended for (i) detection of plant DNA; (ii) plant species determination (soybean, maize, potato, rice); (iii) identification of transgenic elements, including 35S CaMV, 35S FMV, rice actine gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, nptII marker genes. The limit of detection was 0.5% of genetically modified (GM) soybean and maize in analyzed samples. Identification of transgenic DNA in food and feed products using either the developed approach or real-time PCR led to virtually identical results. The assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of the GM component based on the identified transgene.     

Impact of genetic structures on haploid genome-based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications

Real-time Polymerase Chain Reaction (PCR) based assays are widely used to estimate the content of genetically modified (GM) materials in food, feed and seed. It has been known that the genetic structures of the analyte can significantly influence the GM content expressed by the haploid genome (HG) % estimated using real-time PCR assays; this kind of influence is also understood as the impact of biological factors. The influence was first simulated at theoretical level using maize as a model. We then experimentally assessed the impact of biological factors on quantitative results, analysing by quantitative real-time PCR six maize MON 810 hybrid kernels with different genetic structures: (1) hemizygous from transgenic male parent, (2) hemizygous from transgenic female parent and (3) homozygous at the transgenic locus. The results obtained in the present study showed clear influences of biological factors on GM DNA quantification: 1% of GM materials by weight (wt) for the three genetic structures contained 0.39, 0.55 and 1.0% of GM DNA by HG respectively, from quantitative real-time PCR analyses. The relationships between GM wt% and GM HG% can be empirically established as: (1) in the case of the presence of a single GM trait: GM HG% = GM wt% × (0.5 ± 0.167Y), where Y is the endosperm DNA content (%) in the total DNA of a maize kernel, (2) in the case of the presence of multiple GM traits: GM HG% = N × GM wt% × (0.5 ± 0.167Y), where N is the number of GM traits (stacked or not) present in an unknown sample. This finding can be used by stakeholders related to GMO for empirical prediction from one unit of expression to another in the monitoring of seed and grain production chains. Practical equations have also been suggested for haploid copy number calculations, using hemizygous GM materials for calibration curves.     

抗虫玉米CM8101定性检测方法的建立

转基因抗虫玉米CM8101是经人工改造合成的转Bt抗虫基因Cry1Ab?Ma转基因玉米新品系,由我国自主研发,具有更优良的抗虫性,目前已进入生产性试验阶段,具有广阔的产业化应用前景。依据CM8101的5′端旁侧序列信息,设计并筛选出最佳引物探针组合,通过普通PCR和实时荧光PCR技术,建立了转基因抗虫玉米CM8101的两种特异性定性检测方法。结果显示,普通PCR检测方法检出限达0.1%,实时荧光PCR检测方法检出限达0.05%。这两种定性检测方法的建立为今后准确高效检测转基因抗虫玉米CM8101及其产品提供了新的参考方法。     

Multiplex event-specific qualitative polymerase chain reaction for detecting three transgenic rice lines and application of a standard plasmid as a quantitative reference molecule

The three most well-known genetically modified (GM) rice lines in China are TT51-1, KMD1, and KF6. The purposes of this study were to establish a multiplex event-specific qualitative polymerase chain reaction (meqPCR) system for simultaneous detection of the three transgenic rice events and to construct a plasmid as the reference molecule for quantitative analysis. Event-specific primers for each event were selected or designed by focusing on the transgene borders between the inserted DNA and the flanking rice DNA. The developed meqPCR was anticipated to detect distinct amplicons as 454, 398, 301, and 250 bp from KF6, KMD1, TT51-1, and the rice endogenous reference gene, respectively. The robustness of the meqPCR was tested with different levels of the three transgenic rice genomic DNAs, and the sensitivity threshold of the meqPCR was at least 50 ng of 0.1% rice DNA for each event when the three transgenic rice events present and with other GM materials together. The constructed plasmid was evaluated using mixed samples with known GM contents in real-time quantitative PCR. The results indicated that the constructed plasmid was acceptable and suitable for GM rice quantitative analysis.