Absolute quantitative detection of genetically modified soybean MON87708×MON89788 with stacked traits by digital polymerase chain reaction

The main advantage of digital PCR (dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve. Crystal droplet dPCR has a three-color staining detection function, which enables multiplex PCR reaction. In this study, this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 ​× ​MON89788 with stacked traits. Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708 ​× ​MON89788 soybean. Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.     

转基因大豆中外源基因g10evo-epsps 的qPCR检测方法的建立和验证

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。     

Event-specific qualitative and quantitative PCR detection of MON863 maize based upon the 3′-transgene integration sequence

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.     

转基因玉米MON87427/zSSIIb 二重微滴数字PCR方法建立及应用

实施转基因产品定量标识制度需要建立准确可靠的定量检测技术和方法。数字PCR（dPCR）不依赖标准 物质，实现对DNA分子的绝对定量，已成功用于转基因含量检测和标准物质定值。为建立可靠的dPCR方法，获得 准确测量结果，本研究以耐除草剂玉米MON87427为材料，探索建立二重微滴数字PCR（ddPCR）的策略。以构建的 聚合MON87427转化体和5个玉米内标基因的重组质粒pUC57-M为质控样品，将5个不同的玉米内标基因分别与 MON87427组合，通过优化退火温度，根据阳性微滴与阴性微滴分辨率、中等信号强度雨滴数量及测量值与理论值 的一致性等，确定了二重ddPCR组合为MON87427/zSSIIb，最适退火温度为58.4℃。MON87427/zSSIIb 二重ddPCR的 动力学范围为10～60 000拷贝。对盲样进行定量检测，二重ddPCR的定量结果与荧光定量PCR有良好的可比性， 表明MON87427/zSSIIb 二重ddPCR可取代qPCR方法进行转基因玉米MON87427的定量检测及标准物质定值。     

应用单管巢式和半巢式PCR检测转基因玉米MON89034

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。     

进口转基因大豆品系检测及分析

用17种大豆品系特异性检测方法对我国进口自美国、巴西、加拿大和阿根廷四个国家的41批转基因大豆进行检测。分析检测结果显示:在41批进口大豆中仅17个品系中的7种批准大豆品系被检出,分别是MON89788、GTS40-3-2、MON87701、MON87708、A2704-12、A5547-127和FG72,检出率分别为90.24%、87.80%、43.90%、41.46%、36.59%、17.07%和2.44%;不同批次样品检出品系不同,其中6批样品检出1种品系,其余样品均能检出2~5种品系;各国大豆检出品系也不同,其中美国检出7种,而巴西、加拿大和阿根廷分别检出6、5和3种;尽管不同国家大豆检出品系和含量不同,但4大转基因大豆出口国均有GTS40-3-2和MON89788两种品系检出,且含量高。期待以上结果为我国进境转基因大豆检测提供参考,并作为进口转基因大豆安全监管的依据。     

A specific qualitative and real-time PCR detection of MON863 maize based on the 5′-transgene integration sequence

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.     

应用多重荧光PCR快速筛查作物中转基因成分研究

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。     

706份常规大豆品系转基因成分筛查及检测

为调查分析我国的常规大豆育种材料中是否混入转基因大豆品系,选取706份大豆品系,对可能含有的 外源转基因调控元件（CaMV35S启动子、FMV35S启动子和NOS终止子）以及针对我国批准进口的主要耐除草剂转 基因大豆转化体（GTS40-3-2和MON89788）成分进行PCR扩增,同时根据MON89788转化体侧翼序列和目的基因 设计引物做特异性检测,为其定性检测提供技术支撑。结果表明,在选取的所有样品中,最终测得含有CaMV35S启 动子的转基因成分所占比例为1％,含有FMV35S启动子的转基因成分所占比例为0.85％,含有NOS终止子的转基 因成分所占比例为0.71％,耐除草剂转基因品系MON89788所占比例为0.85％,以此评估转基因大豆所占比例,为 农业转基因监管部门开展转基因大豆监管工作提供数据支持。     

A specific qualitative and real-time PCR detection of MON863 maize based on the 5′-transgene integration sequence

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.     

耐除草剂转基因玉米CM8401的获得及功能性状鉴定

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。     

转基因耐除草剂玉米C0010.2.2定性PCR检测方法的建立

转基因耐除草剂玉米C0010.2.2是北京大北农生物技术有限公司利用农杆菌介导法,将epsps基因和pat基因转到受体玉米DBN567获得的耐除草剂玉米转化体,具有耐除草剂草甘膦和草铵膦性状。研究建立转基因耐除草剂玉米C0010.2.2的检测方法,在外源基因插入位点的左、右边界分别设计引物,经过引物筛选、特异性测试、灵敏度测试、退火温度和引物浓度测试,建立转基因耐除草剂玉米C0010.2.2的定性PCR检测方法,该方法的检出限和灵敏度可达到0.1%。验证结果表明,该方法可以特异性检测到转化事件,具有很好的重复性和再现性。     

Low susceptibility of Spodoptera cosmioides,Spodoptera eridania and Spodoptera frugiperda (Lepidoptera: Noctuidae) to genetically-modified soybean expressing Cry1Ac protein

Spodoptera cosmioides (Walker), Spodoptera eridania (Stoll) and Spodoptera frugiperda (J. E. Smith) have caused significant damage on soybean Glycine max (L.) Merrill in Brazil. Genetically-modified MON 87701 × MON 89788 soybean that expresses the Cry1Ac protein is potentially an alternative tool for the management of these species. Purified protein bioassays were done to evaluate the susceptibility of S. cosmioides, S. eridania and S. frugiperda to Cry1Ac protein. The level of efficacy of the Bt soybean plants in controlling these species was measured through laboratory and greenhouse trials under high artificial insect infestations. The biology of these insects was evaluated over their development cycles to understand their life history when fed on Bt soybean. Purified Cry1Ac protein at the maximum concentration tested (100 μg Cry1Ac mL⁻¹ diet) resulted in low mortality of S. cosmioides and S. eridania (<13%) and intermediate mortality of S. frugiperda (50%). No significant effects of the Bt soybean plants were observed in the life table parameters of S. cosmioides and S. eridania. However, S. frugiperda fed on Bt soybean plants had a prolonged larval stage (by 5 days), reduced larvae viability, increased mean generation time (by 8 days) and reduced intrinsic rate of increase. In general, the Bt soybean plants showed poor control of Spodoptera species when evaluated by leaf-disc bioassay and greenhouse trials. Consequently, other control tactics must be used in combination with MON 87701 × MON 89788 soybean in the field for the efficient management of S. cosmioides, S. eridania and S. frugiperda.     

基因组DNA快速提取及在转基因大豆检测中的应用

基因组DNA的提取是DNA分子水平研究和检测的重要环节。为补充完善现场检测方法,根据硅膜吸附 DNA的特性,结合过滤膜和注射器,开发一种现场快速提取植物基因组DNA的方法。选取大豆、棉花、油菜、玉米、 水稻5种主要作物的叶片和种子为样品提取DNA,利用PCR和普通重组酶聚合酶扩增（recombinase polymerase am⁃ plification, RPA）对快速提取和QIAGNE试剂盒提取的DNA进行内源基因的扩增。结果显示,快速方法不需要离心 机,现场3分钟完成DNA提取;在DNA得率上要高于QIAGEN方法,但在DNA纯度上低于QIAGEN方法。不过两种 方法提取的DNA都能满足PCR和RPA的需要。利用DNA快速提取方法结合普通PCR、荧光RPA和荧光定量PCR 对转基因大豆SHZD32-1进行实际检测,3种方法的检测结果一致,均能准确检出大豆SHZD32-1的转基因成分。     

转基因玉米特异性检测阳性标准分子的构建与应用

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。     

广州市农贸市场中转基因大豆的检测

对广州市农贸市场中销售的大豆进行转基因大豆的初步筛查,以检测在广州市场是否有转基因大豆流通和销售,为相关标识和监管提供实验依据。根据转基因大豆内参凝集素Lectin基因及外源基因CaMV35S、NOS及EPSPS基因序列设计4对引物,对收集的大豆样品提取其基因组DNA后进行PCR检测。结果在所收集的14份大豆样品中检测出1份转基因大豆,表明在广州农贸市场中存在转基因大豆,并在市场流通,提醒相关部门需根据国家规定需要加强监管力度。     

中国转基因大豆进口及其影响分析

为明晰转基因大豆进口对中国的大豆产业、国际贸易地位以及转基因发展的影响,进而为壮大国内大豆产业,稳定国内大豆市场秩序,促进国内转基因技术有序发展提供相关依据,利用文献综述方法并结合分析相关统计数据,对我国转基因大豆进口增加的现状与原因进行了阐述。研究认为,转基因大豆进口严重弱化了中国在国际大豆贸易中的话语权,不利于中国的大豆甚至粮食安全以及国产大豆的发展;国际资本伴随大豆进口入侵中国大豆加工产业,同时刺激内资企业的兴起;转基因大豆进口对种质资源、生态坏境存在潜在压力,但又促进国内转基因技术的发展以及转基因监管体系的完善。鉴于此,提出提高国产大豆的市场价值与比较收益,降低国内对国际大豆的依赖度,以需求拉动国产大豆供给,以及在风险可控的情况下加快国内转基因技术研究及监管体系建设的对策建议。     

对引进的美国大豆品种进行转基因成分的检测

利用田间表型鉴定和PCR检测相结合的方法,对从美国引进的46个大豆品种进行了转基因成分的检测.实验过程中,首先通过喷洒草甘膦对待测材料进行表型鉴定,然后通过PCR分子检测分析CaMV35S启动子和NOS终止子的有无,初步判定引进材料是否为转基因大豆,最后对目的基因CP4-EPSPS进行检测,进一步确定所测材料是否为抗草甘膦转基因大豆.大豆本身的凝集素基因作为PCR检测的内置检测标准,有效排除了由于DNA质量、实验操作等因素对检测结果的影响.结果表明,在所检测的46个引进品种中,5个是抗草甘膦转基因品种,41个是非转基因品种,它们可作为我国大豆新品种选育的亲本材料.     

Antibacterial finishing of cotton fabrics using biologically active natural compounds

The paper discusses a method to functionalize cotton fabrics using biologically active natural compounds to achieve the antibacterial characteristics required for medical application. The biologically active natural compounds include propolis, beeswax, and chitosan. Three 100 % cotton knitted fabrics with different degrees of compactness were impregnated in the emulsions containing the active ingredients and fabric variant G3 with the highest degree of impregnation was considered for the evaluation of the antibacterial properties and comfort characteristics. The results show that the treated cotton fabric had high antibacterial activity against both gram positive bacteria Staphylococcus aureus and Streptococcus β haemolytic, and gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. The presence of the biologically active natural compounds on the cotton substrates modified the surface of the textile fibers as seen in the SEM images. The treatment also improved fabric comfort properties, the cotton substrates became less air permissive and more hygroscopic after the treatment. The experimental results indicated that propolis, beeswax and chitosan can be applied as an emulsion to functionalize cotton textile materials. The antibacterial performance of the functionalized fabrics suggested that the cotton fabrics treated with those biologically active natural compounds have the potentials to be used in medical fields.     

Relative Fitness of Helicoverpa armigera (Lepidoptera: Noctuidae) on Seven Host Plants: A Perspective for IPM in Brazil

The cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is a widespread pest of many cultivated and wild plants in Europe, Africa, Asia, and Australia. In 2013, this species was reported in Brazil, attacking various host crops in the midwestern and northeastern regions of the country and is now found countrywide. Aiming to understand the effects of different host plants on the life cycle of H. armigera, we selected seven species of host plants that mature in different seasons and are commonly grown in these regions: cotton (Gossypium hirsutum, “FM993”), corn (Zea mays, “2B587”), soybean (Glycine max, “99R01”), rattlepods (Crotalaria spectabilis), millet (Pennisetum glaucum, “ADR300”), sorghum (Sorghum bicolor, “AGROMEN70G35”), and cowpea (Vigna unguiculata, “SEMPRE VERDE”). The development time of immatures, body weight, survivorship, and fecundity of H. armigera were evaluated on each host plant under laboratory conditions. The bollworms did not survive on corn, millet, or sorghum and showed very low survival rates on rattlepods. Survival rates were highest on soybean, followed by cotton and cowpea. The values for relative fitness found on soybean, cotton, cowpea, and rattlepods were 1, 0.5, 0.43, and 0.03, respectively. Survivorship, faster development time, and fecundity on soybean, cotton, and cowpea were positively correlated. Larger pupae and greater fecundity were found on soybean and cotton. The results indicated that soybean, cotton, and cowpea are the most suitable plants to support the reproduction of H. armigera in the field.