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。     

转基因大豆MON89788双重数字PCR通用定量检测方法的建立

建立一种特异、稳定、灵敏、通用的转基因大豆MON89788品系双重数字聚合酶链式反应（digital polymerase chain reaction,dPCR）定量检测方法,在一个体系内同时进行内外源基因的定量检测,适用于微滴式数字PCR（droplet digital PCR,ddPCR）和芯片式数字PCR（chip digital PCR,cdPCR）平台,并通过了食品分析能力评价体系国际能力验证项目的盲样检测评价。ddPCR平台对大豆MON89788品系和内源Lectin的绝对定量限分别为8.0copies/μL和8.2copies/μL;cdPCR平台对大豆MON89788品系和内源Lectin的绝对定量限分别为7.443copies/μL和7.646copies/μL;ddPCR和cdPCR对转基因大豆MON89788品系成分相对含量的相对定量限均为0.1%。     

转基因苜蓿草J101品系特异性定性PCR检测 方法的建立

目的建立转基因苜蓿草J101品系特异性定性PCR检测方法。方法根据转基因苜蓿草品系J101 5’端外源插入片段与苜蓿草基因组DNA之间的邻接区序列设计引物,建立了转基因苜蓿草J101品系特异性定性PCR检测方法,并对本方法的特异性、灵敏度进行了测定。结果建立的检测方法特异于转基因苜蓿草J101检测,检测最低DNA浓度为(1imit of detection,LOD)为80 pg,相当于50拷贝转基因苜蓿草J101基因组DNA。结论本研究建立的转基因苜蓿草J101品系特异性定性PCR检测方法特异性好,灵敏度高,能够快速、准确地对转基因苜蓿草J101进行检测分析。     

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。     

土壤磷素高效利用转基因大豆特异性PCR检测方法

华南农业大学根系生物学研究中心采用拟南芥的紫色酸性磷酸酶基因AtPAP15转化大豆品系粤春03-3（YC03-3）,获得了酸性磷酸酶活性明显提高、可高效利用土壤磷素的转基因大豆新品系AP15-1。本研究以AP15-1为研究对象,应用TAIL-PCR技术,根据载体序列设计特异引物,获得了转化载体左侧插入的旁邻序列。设计事件特异性检测引物,进行PCR扩增,只能在AP15-1的样品中扩增出特异性条带,进一步用实时荧光定量PCR作分析,结果显示,该引物对重复性好,融解曲线显示只有一个特异峰值。本实验应用该引物对建立的检测方法,检测的灵敏度可以达到0.01%,实时荧光定量PCR检测的极限值可以达到9个基因组的拷贝数,能够满足对转基因大豆新品系AP15-1及其衍生品种检测的需要。     

环介导等温扩增法检测转基因玉米MON89034

根据MON89034外源插入片段与植物基因组序列设计特异性引物,筛选最佳引物并对反应体系和反应条件进行优化,最终建立转基因玉米MON89034转化体特异性LAMP检测方法。对该方法进行了特异性、灵敏度、稳定性和重复性测试。结果表明:该方法能够特异性检测出MON89034玉米;检测其灵敏度达到1 pg;以转基因玉米MON89034 DNA标准品质量分数为1.00%,0.10%,0.05%的样品为模板,其稳定性好、重复性高,假阴性率为0。本试验设计的LAMP方法适用于特异性检测转基因玉米MON89034。     

LAMP实时浊度法快速检测转基因玉米MON810

LAMP实时浊度法是采用环介导等温扩增（Loop Mediated Isothermal Amplification,LAMP）技术通过实时浊度仪实时检测反应过程中所产生的白色沉淀,从而实现对扩增全过程的监控,弥补了显色法只能观看反应终点的缺陷,使引物筛选和反应体系的优化有数据可依。本研究以转基因玉米MON810为研究对象,针对外源基因苏云金芽孢杆菌杀虫毒蛋白CryIA(b)与内源基因边界序列设计6条特异性引物,通过实时浊度法在63 ℃的恒温条件下完成检测,对检测的灵敏度、特异性、稳定性进行了评价。建立了转基因玉米MON810的LAMP实时浊度检测方法,该方法最低检出限为0.5%,与LAMP显色法和实时荧光PCR法进行结果比对,符合率为100%,经评价具有特异性高、稳定性强、准确简便等优点,非常适合转基因玉米MON810的快速检测,有较好的应用价值。     

转基因食品的定量PCR检测方法

近年来转基因食品定性检测方法发展迅速,然而目前转基因成分(GMO)的准确定量检测在国际贸易中日趋重要.我们这里介绍三种定量检测方法半定量PCR法,定量竞争PCR(QC-PCR)法和实时定量PCR法.半定量PCR法比较简单,但结果不是很精确.定量竞争PCR的特点是含有内部标准子.近来开展的实验室合作研究表明,与定性PCR法相比,定量竞争PCR降低了实验室之间的误差.而实时定量PCR法可在提取DNA后3h内,测出每克起始样品的总DNA量及2pg转基因成分的量,但这套PCR系统目前价格昂贵.     

转基因食品定量检测方法的验证

按欧盟联合研究中心(Joint Reaserch Center,JRC)的欧盟网络转基因实验室(European Network of GMO laboratories,ENGL)方法验证工作组编制的指南文件的要求,对《GB/T 19495.5—2004转基因产品检测核酸定量PCR检测方法》进行方法验证。分别验证了脱氧核糖核酸(deoxyribonucleic acid,DNA)提取质量,方法的准确度、重复性和相对定量限4个指标。经过验证,选用的DNA提取试剂盒提取质量合格;检测值在可接受参考值的±25%内,准确度良好;相对可重复性标准偏差均小于25%,重复性良好;相对定量限为0.1%转基因含量。依照指南对国标进行了验证,结果证明该指南在实验室进行方法验证中具有指导性作用。     

转基因苜蓿草J163品系特异性实时荧光PCR检测方法的建立

目的建立针对我国农业部未颁发农业转基因生物安全证书的转基因苜蓿草品系J163品系特异性实时荧光(Polymerase Chain Reaction,PCR)检测方法。方法利用Taq Man实时荧光PCR(real-time PCR)技术,根据转基因苜蓿草品系J163 5’端外源插入片段P-e FMV与苜蓿草基因组DNA之间的邻接区序列设计引物和探针,建立了转基因苜蓿草J163品系特异性实时荧光PCR检测方法,并对本方法的特异性、灵敏度及可重复性进行了测定。结果建立的检测方法特异于转基因苜蓿草J163成分检测,检测最低DNA浓度为(1imit of detection,LOD)为15 pg,相当于9拷贝转基因苜蓿草J163基因组DNA,重复性试验显示,其标准偏差(Standard deviation,SD)和相对标准偏差(relative standard deviation,RSD)均在可接受范围内。结论本研究建立的转基因苜蓿草J163品系特异性实时荧光PCR检测方法特异性好,灵敏度高,能够快速、准确、稳定地对转基因苜蓿草J163成分进行检测分析。     

Development and in‐house validation of the event‐specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985

BACKGROUND: To implement genetically modified organism (GMO) labeling regulations, an event‐specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. RESULTS: In this study, specific primers and TaqMan probes based on the revealed 5′‐end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg^?1 in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in‐house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in‐house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. CONCLUSION: This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates. Copyright © 2009 Society of Chemical Industry     

Event-specific qualitative and quantitative PCR detection of genetically modified rapeseed Topas 19/2

The herbicide-tolerant transgenic rapeseed Topas 19/2 (synonym HCN92) has been approved for environmental release in Canada, Japan, Australia and the USA, and exported to a number of other countries as raw material. The purpose of this study was to establish event-specific qualitative and quantitative detection methods for Topas 19/2. The 3′-integration junction sequence spanning the host plant DNA and the integrated transgene of the Topas 19/2 event was isolated and identified. The event-specific qualitative detection method was established to produce an amplicon of 110 basepairs (bp) with an absolute detection limit of 10 initial template copies. The event-specific quantitative detection method was developed with the limit of detection (LOD) and limit of quantification (LOQ) being approximately 5 and 50 initial template copies, respectively. The developed real-time PCR systems were assessed using two mixed rapeseed samples with known Topas 19/2 contents. Expected results were obtained.     

转基因鲑鱼AquAdvantage品系特异性实时荧光聚合酶链式反应检测方法的建立

目的实现转基因鲑鱼AquAdvantage的标识管理,建立其品系特异性实时荧光聚合酶链式反应(PCR)检测方法。方法针对转基因鲑鱼的品系特异性序列设计引物和TaqMan探针,建立转基因鲑鱼实时荧光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。结果建立的转基因鲑鱼实时荧光PCR方法特异性强,在600 000~60拷贝范围内呈良好的线性关系,其线性回归方程为y=-3.2194x+40.805,R~2=0.997,检测限为60拷贝,检测重复性良好。结论建立的品系特异性实时荧光PCR方法可应用于转基因鲑鱼AquAdvantage的鉴定。     

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。     

烟草转基因内标准定量检测中竞争性模板的制备

以转基因烟草为材料，通过DNA提取、PCR扩增获得NOS终止子片段产物，以重叠延伸扩增法制备该片段的竞争性模板，并进行酶切验证。     

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.     

Establishment and evaluation of event-specific quantitative PCR method for genetically modified soybean MON89788

A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C(f)) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON89788.