多品系混杂转基因大豆样品中复合品系的检测

摘要：目的 确认一份多品系混杂转基因大豆样品中是否含有复合性状转基因大豆。方法 采用实时荧光定性PCR方法对实验室留存的一份转基因初筛阳性大豆样品进行品系筛查检测,后分别采用单粒多靶点筛查检测和清洗后单粒多靶点筛查检测方法进行复合性状品系的确认。结果 经鉴定,该大豆样品中混杂了5种转基因大豆品系,检出复合性状转基因大豆为MON87708×MON89788品系,该品系为中国未经批准进口的转基因大豆品系。结论 鉴于目前缺乏完整的检测技术体系和技术标准,这种多品系混杂样品中掺杂未批准复合性状品系的行为非常具有隐蔽性和欺骗性,国内的农业安全监管部门和海关检疫部门都应该对此高度重视。     

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。     

大豆及豆制食品中转基因成分检测分析

建立大豆及豆制食品中转基因成分分析的real-time PCR扩增体系和方法。选择市售的大豆、豆粉、豆干、豆腐、豆浆和腐竹等6种大豆及豆制食品,针对不同的样品探索高质量基因组DNA的提取方法,扩增大豆内参基因lectin进行质量检测。针对我国批准的3种主要转基因大豆品系(GTS40-3-2,A2704-12和MON89788)设计特异性的引物和探针,进行real-time PCR检测。购买3种转基因大豆品系的标准品作为质控对照。从该6种大豆及豆制食品中均提取到了高质量的基因组DNA,建立了稳定的针对外源基因特异性、结构特异性和品系特异性片段检测的real-time PCR扩增体系。成功建立了6种大豆及豆制食品中针对3种转基因大豆品系成分检测的real-time PCR方法。     

实时浊度LAMP法检测豆制品中转基因成分

通过设计转基因大豆内源基因(Lectin)、外源基因NOS和2个转基因大豆品系(GTS 40-3-2,MON89788)特异性LAMP引物,建立快速检测转基因大豆以及鉴定转基因大豆品系的LAMP体系,并选取国内豆制品利用CTAB法进行DNA提取,并针对上述基因进行PCR和LAMP扩增的对比,建立起了检测大豆制品中转基因成分的定性的LAMP方法。结果表明:4个LAMP方法均有较好的特异性,除了NOS、GTS 40-3-2的检测限为0.1%外,其他LAMP方法的灵敏度为0.01%,均能满足实际检测要求;在实时浊度LAMP法检测大豆制品转基因成分的鉴定中,传统的CTAB法能成功提取大豆粗加工制品的DNA,且DNA纯度能够进行核酸扩增反应。利用转基因外引物进行的普通PCR结果与实时浊度LAMP结果是一致的,说明建立的转基因实时浊度LAMP检测方法有很好的准确性。     

烘焙食品中转基因成分定性检测的FAPAS能力验证结果与分析

目的分析参加FAPAS烘焙食品中转基因成分定性检测能力验证的结果。方法根据FAPAS组织方提供的能力验证作业指导书、国家标准GB/T19495.3-2004转基因产品检测核酸提取纯化方法(部分步骤改进)和行业标准SN/T1204-2016《植物及其加工产品中转基因成分实时荧光PCR定性检验方法》对一份烘焙食品测试样品进行转基因成分定性检测。结果该样品检出pCaMV35S、tNOS、GTS40-3-2和MON863品系(基因),未检出Bt11、Bt176、GA21、MIR604、MON810、MON88017、MON89034、MON89788、NK603和TC1507品系。结论本实验重点对核酸提取方法进行改进并严格质控,满足了烘焙食品这类加工样品转基因定性检测要求,获得能力验证结果为满意。     

公共引物介导的多重定量PCR技术在转基因大豆检测中的应用研究

目的 建立公共引物介导的多重定量PCR法检测我国农业农村部允许进口的6种转基因大豆(包括: MON87701、DP356043、CV-127、MON87769、MON87705、MON87708)的方法。方法 设计并筛选出针对本研究涉及的6种转基因大豆对特异性引物, 在每对特异性引物的5’端均加上一段公共引物, 形成特异性嵌合引物。以某种转基因大豆DNA为模板, 加入多引物体系以及荧光公共引物验证GeXP(gene expression platform)多重检测体系的特异性, 每种转基因大豆DNA模板浓度分别稀释为20、2、1、0.2、0.02 ng/μL, 运用GeXP多重PCR方法进行单一DNA模板灵敏度分析并优化各对特异性嵌合引物的工作浓度。结果 多重PCR检测体系扩增出特异性片段, GeXP检出的各转基因大豆片段长度MON87701、DP356043、CV-127、MON87769、MON87705和MON87708分别为140.75、184.42、274.93、308.47、467.98和517.67 bp, GeXP 多重检测体系检测敏感性可达到为1 ng/μL, 扩增产物测序结果进一步说明了引物扩增的特异性。结论 本研究建立的基于GeXP系统的多重PCR定量检测技术, 可以准确鉴别6种转基因大豆, 为转基因大豆及其他转基因产品提供了新型的检测方法。     

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%。     

改良LAMP法检测转基因作物

目的 建立简易的可视化环介导等温扩增(LAMP)检测转基因作物的方法。方法 通过一对特异的能识别一段正义链和一段反义链的内引物和一对链置换引物, 在Bst DNA聚合酶作用下, 以等温条件实现对外源转基因成分的扩增, 反应液和高浓度的显色液SYBR GREENⅠ同时置于特殊设计的反应管中, 反应后可实现闭管可视化检测。结果 对转基因作物中常见的外源基因元件胭脂碱合成酶终止子(T-NOS)和烟草花叶病毒35S启动子(P-CaMV35S)进行了LAMP检测。T-NOS LAMP对大米品系BT63, 玉米品系BT11, MON810, 大豆品系GTS40-3-2的检测结果呈阳性, P-CaMV35S LAMP对大米品系BT63, 玉米品系BT11, MON810, MON863, 大豆品系GTS40-3-2的检测结果呈阳性, 二者对非转基因大米, 玉米, 大豆的检测结果均呈阴性。上述结果表明, 本文中建立的T-NOS 和P-CaMV35S LAMP检测方法特异性高。另外, 通过对大豆品系GTS40-3-2模拟样品的检测, 证明T-NOS LAMP能检出转基因成分含量为0.5%的样品, P-CaMV35S LAMP能检出转基因成分含量为0.1%的样品, 检测灵敏度较高。结论 改良LAMP法灵敏度高, 特异性好, 能用于快速初筛转基因作物。     

Fapas大豆粉中转基因成分检测能力验证结果与分析

目的提升实验室检验人员的检验水平,扩大实验室影响力,增强竞争力。方法根据Fapas组织方提供的能力验证作业指导书及GB/T19495.4-2004《转基因产品检测核酸定性PCR检测方法》和SN/T1204-2016《植物及其加工产品中转基因成分实时荧光PCR定性检验方法》对一份100%大豆粉测试样品进行转基因成分检测。结果该批次大豆粉样品pCaMV35S、tNOS、Roundup Ready(GTS-40-3-2)品系检出转基因成分, MON89788品系未检出转基因成分。结论本次能力验证结果为满意。     

进口油菜籽中不同转基因品系的检测与分析

对我国27批进口油菜籽进行了转基因成分检测和转基因油菜品系组成情况分析。利用已知转基因油菜RT73、T45、Oxy235、Topas19/2、MS1、RF1、RF2、MS8和RF3的特异性检测体系,对初检为阳性的转基因油菜籽样品进行扩增,扩增基因包括bar、PAT、GOX和NPTII。结果共检出25批转基因油菜籽,检出率为92.6%。所有批次均检出品系RT73和MS8×RF3,表明二者为目前我国进口的主要转基因油菜籽。约40%的批次检出MS1×RF1和MS1×RF2,有少量批次检出T45、Oxy235和Topas19/2,每批次均为多品系混合样品。研究表明,列入农业转基因生物安全生产证书（我国农业部颁发）名单的7个转基因油菜品系均有进口,其中以RT73和MS8×RF3为主。     

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.     

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.     

Multiplex real-time PCR for the detection and quantification of DNA from four transgenic soy Mon89788, A5547-127, Roundup Ready, A2704-12 and lectin

In the past, the transgenic soybean Roundup Ready was the predominantly cultivated transgenic soybean. With the announcement of Monsanto Corp. that this trait will be replaced by Mon89788 and the release of other company??s transgenic soy crops, this unique status of one trait has ended. Therefore, a multiplex quantitative real-time PCR system was developed and characterized for three additional transgenic traits Mon89788, A2704-12 and A5547-127. It showed amplification efficiency, correlation and sensitivity similar to the single PCR systems applied therein. This system allows relative multiplex quantification and/or delta?Cdelta Ct method and therefore an efficient control of products during production, trade and sale.     

Multiplex real-time PCR Method for the detection and quantification of DNA from the four transgenic soy traits DP-356043-5, DP-305423-1, MON 87701, and BPS-CV127-9 and lectin

According to the release of authorization, several new transgenic soy crops are expected to be present in the harvest of soy beans. In contrast to earlier times, methods for the detection of these transgenic crops are available already in advance. However, these methods are mainly still inefficient single real-time PCR methods. To increase the efficiency of product control, a multiplex quantitative real-time PCR system was developed and characterized for the four new transgenic soy traits DP-356043-5, DP-305423-1, MON 87701 and BPS-CV127-9. It showed amplification efficiency, correlation and sensitivity similar to the single PCR systems applied therein. To evaluate the robustness, an appropriate testing scheme was developed and applied for the first time on this multiplex real-time PCR system. It showed the robust amplification of all analytes also in case where conditions were varied. This system allows relative multiplex quantification and/or delta–delta Ct method quantification and proofed the applicability in routine.