LAMP实时浊度法检测转基因水稻Bt63品系

采用环介导等温扩增技术(LAMP),针对转基因水稻Bt63品系外源基因与内源基因结合处序列设计4条特异性引物,利用浊度仪实时监测扩增过程,建立Bt63品系的快速检测技术体系,并对反应温度、灵敏度、特异性、稳定性和实际样品检测能力进行初步探索。结果表明,LAMP实时浊度法能够特异性检测转基因水稻Bt63品系,其最低检出限为0.01%,是普通PCR方法的10倍,具有广泛的应用价值。     

转Bt基因水稻的食用安全性评价研究进展

水稻（Oryza sativa）是我国重要的经济作物,其优化育种一直是研究的重点。苏云金芽孢杆菌（Bacillusthuringiensis,Bt）抗虫基因的出现,开辟了利用外源基因培育转基因抗虫作物的新领域。目前,转基因抗虫水稻的培育技术已日趋成熟,而其对人体的潜在安全性问题却备受争议。转Bt基因水稻能否实现商业化是人们关注的焦点。本文就转Bt基因水稻的食用安全性评价的研究进展进行综述,以期为我国转Bt基因水稻的风险评估和推广提供一些参考。     

转Bt基因水稻种子中Bt蛋白含量测定方法的研究

采用ELISA技术检测转Bt基因水稻中Bt蛋白的含量,判断水稻样品中是否含有转基因成分。利用研磨、酶标、孵育等技术对样品进行前处理。采用阳性质控物浓度等倍稀释方法,建立标准曲线,相关系数为0.997 4。用一系列不同转基因含量的标准基体材料,分析方法的最低检测限,灵敏度达0.1%。通过对我国进入生产性试验的转Bt基因水稻品系TT51-1和科丰6号的测试,表明该方法与普通PCR方法真实性和灵敏度一致,可以广泛应用于转Bt基因水稻及其粗加工产品的转基因成分检测。为转基因生物安全监管和安全性评价提供技术支撑。     

利用实时荧光定量PCR方法分析转基因水稻外源基因拷贝数

为分析转基因水稻外源基因拷贝数，利用新型、灵敏、高通量的实时荧光定量PCR方法进行转基因水稻外源基因拷贝数的分析。转基因外源基因的拷贝数通过转基因水稻外源基因(GUS和HPT基因)和水稻内标准SPS基因含量的比较计算获得。定量分析了14株T0代的转基因水稻植株，得到了外源基因插入分别为1、2、3和4的转基因植株，同时利用Southern Blot方法进行验证分析。随机选择18个已经过定量PCR检测分析的转基因水稻植株，用Southern Blot的方法分析转基因水稻植株中的HPT或GUS基因的拷贝数，Southern Blot分析结果显示有15个转基因水稻植株的分析结果与定量PCR分析的结果是一致的，3个植株定量PCR分析的转基因拷贝数稍高于Southern Blot的分析结果，主要原因是Southern Blot方法在同一个插入位点有多拷贝的T-DNA片段插入时，转基因植株的基因组在完全酶切时会产生相似的DNA片段，电泳分析时很难分辨清楚。定量PCR方法则完全避免了这种情况的发生，除非目的基因DNA片段在PCR引物处发生断裂。两种方法分析结果的比较显示定量PCR方法分析转基因拷贝数更加有效、适用。     

豌豆铁蛋白转基因水稻纯系的稻米矿质元素及淀粉特性研究

选取已连续7代自交纯化,并结合GUS标记辅助选择,所获得的26份独立豌豆铁蛋白(pea fer-ritin,Fer)转基因水稻纯系,以秀水11为CK,就豌豆铁蛋白基因(fer)导入水稻后,可能产生对稻米Fe含量、其它矿质元素、蒸煮食用品质、淀粉黏滞特性等影响进行研究。结果表明,26份转基因纯系的精米平均总铁含量为10.37μg/g,变幅5.98~31.28μg/g,但仅有4份(即Fer34、Fer 36、Fer 39、Fer 65)显著高于CK(6.46μg/g),其余22份与CK无显著差异;转基因纯系间在其它8种矿质元素含量方面的变异程度以Cu最大,Zn、Ca次之,S、K、Mn、P、Mg等较小,但仅Mn、Cu变化与Fe含量变异分别有显著负、正相关;转基因纯系间在胶稠度、糊化温度、直链淀粉含量等3个重要蒸煮食用品质性状上,也发生了部分变异,分别有7、4和1份纯系与CK显著不同;但在淀粉黏滞性方面,转基因纯系与CK间差异很小,从而推测豌豆铁蛋白基因导入对稻米淀粉结构及其特性影响不大。并就有关豌豆转基因纯系稻米品质变异可能产生原因和外源基因作用等问题进行了讨论。     

重组酶聚合酶扩增技术检测转基因水稻中的Cry1Ab/c基因

重组酶聚合酶扩增技术(RPA)是利用重组酶和单链结合蛋白在常温下协同实现引物与模板的特异结合,以代替传统PCR热循环中的变性和复性过程的新型恒温体外核酸扩增技术。本研究基于RPA技术建立了转基因水稻Cry1Ab/c基因的检测方法,可在37℃恒温条件下快速检测到转基因水稻中的Cry1Ab/c基因,具有较好的特异性,其绝对及相对检测灵敏度分别达到100个拷贝和0.1%(质量分数),适用于基层实验室及现场快速检测转Cry1Ab/c基因水稻及其制品。     

利用实时荧光PCR方法检测转Bt基因大米

应用实时荧光PCR技术对转基因大米进行了定性和定量检测研究.本研究以转基因B163大米为材料,采用TaqMan探针技术,对大米中的内源基因蔗糖磷酸合酶SPS和转基因水稻中普遍存在的外源基因CaMV35S启动子、NOS终止子以及苏云金芽孢杆菌(Bacillusthndngiemis,简写为Bt)杀虫晶体蛋白基因Cry1Ac进行了实时荧光PCR研究,并对外源基因Cry1Ac进行了定量检测和敏感性分析.该实时荧光PCR方法检测结果和常规PCR结果一致,同时不用进行凝胶电泳,更为快速、简便,降低了污染机会,可用于转Bt基因大米的定性和定量检测.     

外源蛋白基因对棉籽营养成分影响的研究

本研究通过对转Bt毒蛋白棉花的各种营养成分、矿物质含量、氨基酸含量以及抗营养因予成分的检测与分析，评价Bt等外源基因对棉籽营养成分的影响，为转基因产品的安全评价提供依据。通过检测证明，转基因棉籽和非转基因棉籽在营养学方面具有实质等同性。     

改良十六烷基三甲基溴化铵-磁珠核酸提取方法及在植物转基因检测中的应用

目的 建立一种快速、高效、便捷的植物基因组DNA提取方法并运用于植物转基因检测。方法 改良传统的十六烷基三甲基溴化铵(cetyltrimethylammonium ammonium bromide, CTAB)方法, 并结合磁珠吸附基因组DNA, 配合核酸自动提取系统提取水稻和加工米粉中的核酸, 荧光聚合酶链式反应(polymerase chain reaction, PCR)检测Bt63大米转基因成分, 并与另外4种提取方法的结果进行对比, 评价提取效果。结果 5种不同方法中CTAB-磁珠法提取的样品基因组DNA浓度和质量最佳, 荧光PCR检测低浓度Bt63转基因成分(0.01%)的Ct值最小, 检测结果最好。结论 该方法可高效快速地提取植物核酸, 在低含量的转基因成分检测中相比其他几种方法具有绝对优势。     

转基因水稻定性PCR检测体系的建立

采用PCR技术检测CaMV35S启动子、NOS终止子、Bt基因,判断水稻样品中是否含转基因成分,建立了稳定快速的转基因水稻定性PCR检测体系.PCR检测的灵敏度达到0.1%,稳定性良好.结果表明:PCR技术检测外源基因是灵敏和准确的,可以广泛地应用到转基因作物及其加工产品的转基因成分检测中.此外,进一步讨论了转基因成分检测中存在的问题、转基因产品标识的必要性和目前转基因检测技术在检测中的问题.     

Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice

Genetically modified rice varieties developed in China are close to approval for agricultural cultivation and production. However, so far no method has been reported for specific detection of transgenic varieties of this crop. In the present study, rice seeds assumed to consist of field-tested Bt rice (‘Anti-pest Shanyou 63’ and ‘Anti-pest Jinyou 63’) were used as reference material to determine transgenic DNA sequences. The transition between the cryIA(b) and cryIA(c) fusion gene and the nopaline synthase terminator (nos) sequence was used to develop a construct-specific real-time PCR based detection method. This Bt rice specific detection system was combined with a recently published quantitative real-time PCR method for the rice-specific (Oryza sativa L.) reference gene gos9. The complete PCR assay for detection of transgenic Bt rice was in-house validated and the limit of quantification was found to be below 0.1% Bt rice relative to the rice content. Application of the PCR assay should allow more precise detection of transgenic rice varieties in imported food products which are so far not approved in the EU.     

Development of an event-specific Real-time PCR detection method for the transgenic Bt rice line KMD1

The present study describes an event-specific quantitative Real-time PCR detection method for the transgenic Bt rice line Kemingdao 1 (KMD1). This rice line which is not approved in any country so far is likely to be approved in China in the near future. The developed primers amplify a DNA sequence spanning the integration site of the genetic construct in KMD1. DNA sequence information of this unknown site necessary for primer design was achieved using SiteFinding-PCR technique. The specificity of the detection method was shown by testing a number of different transgenic and conventional plant varieties (e.g. rice LL 601, LL 62, Bt 63). As alternative to genomic DNA, we developed double target hybrid amplicons as synthetic calibration standards in Real-time PCR. These amplicons contained both one copy of the KMD1 event-specific sequence and one copy of a sequence of the rice reference gene gos9. The limit of quantification (LOQ) of the method was tested to be 0.05%. R. Babekova and T. Funk contributed equally to this paper.     

Detection of genetically modified rice: collaborative validation study of a construct-specific real-time PCR method for detection of transgenic Bt rice

A collaborative trial study has been conducted for validation of an extraction method and a subsequent real-time PCR for detection of a transgenic Bt rice line (‘Bt63’) in rice products originating from China. A total of 17 laboratories participated in the study and each laboratory received 16 coded samples comprising of rice grain flours, rice noodle flours and plasmid DNAs. Of the accepted results all Bt63-positive rice grain samples (0.1 or 0.05% w/w) and all rice noodle samples prepared from marketed rice products were detected correctly. The result demonstrates that ‘Bt63’ rice is detectable even at low relative mass concentrations of 0.05%. The absolute LOD determined with plasmid DNA samples showed to be at least five copies of the ‘Bt63’ target sequence. The data provided in this study show that the method is fit-for-purpose to inspect Chinese rice products for the presence of EU-unauthorised rice lines carrying the ‘Bt63’ construct.     

A cotton‐specific gene,stearoyl‐ACP desaturase,used as a reference for qualitative and real‐time quantitative polymerase chain reaction detection of genetically modified organisms

Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)‐based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl‐ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real‐time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties. Copyright © 2006 Society of Chemical Industry     

亲代大鼠转Bt基因水稻暴露对其子代部分早期生理和神经发育指标的影响

研究亲代大鼠转Bt基因水稻(TT51大米)暴露对其子代部分早期生理和神经发育指标的影响。方法 亲代大鼠随机分为3组,每组雄性15只,雌性30只,分别给予按60%比例掺入市售大米、明恢63大米和TT51大米的饲料,连续喂养70d后同组雄雌大鼠交配并产生子代,孕期和哺乳期各组母鼠继续给予相应受试大米的饲料。亲代大鼠每周称量和记录体重及食物消耗量,同时观察动物生长发育状况,观察和记录亲代雌鼠生殖指标。仔鼠出生后记录0、4、7、14及21d体重,并观察仔鼠部分生理指标发育情况,同时进行听觉惊愕等神经发育指标的测定。断乳后,对各试验组部分仔鼠取脑等主要脏器进行病理检查。结果 TT51大米组与明恢63大米组和市售大米组比较,亲代大鼠一般毒性及生殖指标间差异均无统计学意义;子代部分生理发育指标和神经发育指标差异均无统计学意义;各试验组仔鼠脏器病理检查均未见有意义的变化。结论 未见亲代大鼠转Bt基因水稻暴露对其子代早期部分生理和神经发育指标有不良影响。     

Development of a real‐time multiplex PCR system for the quantitative detection of Chinese GM rice

BACKGROUND: Rice is one of the most important staple food crops for more than half the global population, who rely on it for as much as 80% of their diet. By one estimate, the world population is projected to grow to approximately 11 billion people by the year 2050. So it is a formidable task to meet the future demand. For this reason, breeders make a great deal of effort to produce new rice varieties with traits such as higher yield, improved nutritional content and better resistance to disease and pests, via transgenic biotechnological protocols. Dozens of transgenic rice lines have been developed since the first transgenic rice plant production in the late 1980s. With the rapid approach of transgenic rice commercialisation, it is becoming necessary to develop techniques capable of detecting and quantifying genetically modified (GM) rice. RESULT: Here we describe a method in which transgenic DNA is quantified by amplifying part of the 35S‐CaMV promoter and standardising it against an amplified portion of an endogenous single copy, rice specific gene encoding sucrose phosphate synthase. Both reactions are performed simultaneously in a single tube. Standard calibration curves were developed by diluting DNA extracted from a blend of non‐transgenic (c.v., Nipponbare) and 5% KMD2 transgenic rice. The method was tested for the quantification of the five GM rice events, including KMD2, Wan 21A, GC‐1, H1597 and TR4, which contain the 35S‐CaMV promoter. The coefficient of variation varied from 3.15% to 12.84%, which is up to acceptance criterion over the dynamic range of the method. CONCLUSION: In this study, we successfully applied a multiplex real‐time PCR assay to GM rice, which employed SPS as the endogenous reference gene and the gene regulation element 35S‐CaMV promoter as a GMO marker. The detection limit and limit of quantification is sufficient to comply with all relevant regulations in the EU and worldwide. The detection system could be applied in routine analysis for the quantification of GM rice in food materials, such as instant rice, unpolished rice, rice flours, biscuit powders, and starch. It may prove useful with regard to a robust screening technique of broad utility as transgenic rice enters global commodity markets. Copyright © 2009 Society of Chemical Industry     

Toxicological evaluation of transgenic rice flour with a synthetic cry1Ab gene from Bacillus thuringiensis

Bacillus thuringiensis (Bt) transgenic (KMD1) and non‐transgenic (KMD1′s parental variety Xiushui 11) rice flours were assessed in a 90 day feeding test with rats. KMD1 contained a synthetic cry1Ab gene from Bt, and selection marker genes nptII and hpt linked in tandem. In the≤64 g kg^?1 body weight (BW) dosage range (Bt transgenic rice flour composed 64% of the ingredients of the diet), no adverse effects of Bt rice on rats were observed in terms of animal behaviour, weight gain and feed utilisation rate. Necropsy at the end of the experiment indicated that neither pathological lesions nor histopathological abnormalities were present in organs such as liver, kidneys, intestines and testes of rats in both test and control groups by macroscopic and microscopic pathology. In addition, no significant differences (P > 0.05) were observed in relative organ weights, haemograms and blood indices of rats between test and control groups. Several serum parameters of female rats were found to be significantly different between Bt and non‐Bt diets, but the values of these parameters were still within the normal ranges of values for rats of this age and sex. These results demonstrated that Bt rice flour at a dosage of 64 g kg^?1 BW, Bt toxin and NPTII and HPT proteins have no toxic effects on rats. © 2002 Society of Chemical Industry     

Selection of a Taxon-Specific Reference Gene for Qualitative and Quantitative PCR Detection of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis>

Safflower (Carthamus tinctorius) is an emerging model plant for the transgenic modification of fatty acid composition and the production of pharmaceuticals, proteins, or enzymes. Safflower is also a traditional Chinese medicine and is often used as a fake saffron product. Reliable detection of an endogenous reference gene is indispensable for the supervision of genetically modified safflower. Such an endogenous reference gene can also be used to specifically identify safflower ingredient in complex mixtures such as medicine or food. In this study, we identified and validated the CTOS gene as an endogenous reference for safflower. Conventional and real-time polymerase chain reaction (PCR) methods for detecting the CTOS gene sequence showed high interspecies specificity and intra-species stability. The lowest copy number detectable by conventional PCR was 10 haploid copies. The limit of detection and limit of quantification for the real-time PCR assay were estimated to be five and 40 haploid genome copies, respectively. Standard curves established for the real-time PCR assay exhibited good linearity (R ² > 0.99) between the cycle threshold (Ct) values and the initial template copies. The developed conventional and real-time PCR assays were validated in routine analysis of the safflower ingredient in commercial Chinese medicines. In conclusion, the developed quantitative PCR methods were sufficiently specific and sensitive to be used in safflower genomic DNA quantification and safflower ingredient identification.