大豆和玉米加工产品中外源转基因成分检测技术的建立

目的：建立检测大豆和玉米加工产品中转基因成分的PCR技术.方法：采用PCR技术检测CaMV 35S启动子,并进一步通过PCR检测RoundUp Ready Soybean（RRS）和Bt176 Maximaizer的特异性DNA片段,判断大豆和玉米加工产品中是否含相应转基因成分.结果：在1份豆粕和豆腐样品中检测到了RoundUp Ready大豆特异性的498bp片段,而在玉米粒样品中检测到了Bt176特异性转基因成分.PCR检测的灵敏度达到0.1%,稳定性良好.结论：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.     

PCR法筛选检测转基因食品

目的通过PCR检测花椰菜花叶病毒(camv)35S启动子和胭脂碱合酶(nos)终止子建立筛选食品中有无转基因成分的方法.方法用改良溴化十六烷三甲基铵(CTAB)法制备转基因抗除草剂[大豆RoundUp ReadyTMSoybean(RRS)]和转基因抗虫玉米系列标准品Bt176 Maximaizer的DNA,PCR检测其内参照基因及camv 35 S启动子和nos终止子.结果改良CTAB法制备的DNA用作PCR模板均可扩增出内参照基因,PCR扩增camv 35S启动子可检测出含量为0.5%的RRS和Bt176 Maximaizer,而PCR扩增nos终止子可检测出含1%RRS的食品样品.结论改良CTAB法制备的DNA可用作PCR模板,建立的PCR检测camv 35S启动子和nos终止子方法可用于筛选食品中有无转基因成分.     

玉米加工产品中转基因成分的定量检测

[目的]建立检测玉米加工产品中转基因成分的实时荧光定量PCR技术。[方法]根据转基因Bt176玉米中的外源基因和内源基因设计的引物和TaqMan荧光探针,使用实时定量PCR技术对转基因成分含量进行了定量检测,建立了Bt176玉米参照样品的外源基因和内源基因Ct值之差与转基因成分含量之间的标准曲线和线性回归方程,并对采自市场的未知样品进行了检测。[结果]在1份玉米粒样品中检测到了Bt176玉米转基因成分。检测灵敏度达到0.01%。[结论]实时荧光定量PCR技术检测方法是灵敏和准确的,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。     

SYBR Green Ⅰ实时荧光PCR检测食品中转基因成分方法的灵敏度和特异性研究

目的:研究SYBR Green Ⅰ实时荧光PCR检测食品中转基因成分(35S基因和NOS基因)方法的灵敏度、特异性和线性关系。方法:使用SYBR Green Ⅰ实时荧光PCR方法检测不同转基因含量的标准品,并对转基因阳性和阴性样品分别进行扩增,分析扩增曲线和Ct值。结果:SYBR Green Ⅰ实时荧光PCR检测食品中转基因成分方法检出限为0.05%,转基因大豆标准品和阳性样品中检出35S基因和NOS基因成分,阴性样品中未检出35S基因和NOS基因成分。结论:SYBR Green Ⅰ实时荧光PCR检测35S和NOS转基因成分方法灵敏度高,特异性较好,标准曲线线性良好。     

利用TaqMan定量PCR技术不依赖参比内源基因测定转基因植物外源基因拷贝数

目的:建立一种利用TaqMan荧光PCR定量技术不依赖参比内源基因测定转基因植物外源基因拷贝数的分析方法.方法:以转基因大豆RRS和转基因玉米Bt176为材料,通过转基因植物外源基因与品系特异序列拷贝数的比较测定外源基因整合拷贝数.结果:3次测得的Bt176玉米中Cry1A(b)基因的整合拷贝数分别为2.69,3.43和2.83,平均为2.98.标准偏差(SD)为0.40,相对标准偏差(RSD)为13.28%,即CrylA(b)基因在Bt176玉米中的整合拷贝数为3;3次测得的RRS大豆中EPSPS基因拷贝数分别为1.08,1.01,0.96,平均为1.01,SD为0.06,RSD为6.06%,即RRS大豆中EPSPS基因拷贝数的测定结果是单拷贝.结论:本研究建立的转基因植物外源基因拷贝数测定方法可准确测定转基因植物外源基因的拷贝数.与现有的通过外源基因与参比内源基因拷贝数比较测定转基因植物外源基因拷贝数的方法相比.不但避免了选择物种特异性的看家基因及测定看家基因拷贝数等大量工作,而且应用范围更广泛.     

深圳市场4种国产农产品转基因成分监测结果

目的：为了解并掌握目前深圳市居民食品中转基因成分的实际情况，避免“家底不清”，更有效地对本市的食品安全进行系统监测和预警，为政府制定食品卫生政策提供科学依据和切实保障居民的健康安全利益。方法：按照深圳市食品污染物监测项目方案，首次对本市市场上的4种国产农产品中转基因成分进行监测，采用FCR凝胶电泳法对79份玉米、马铃薯、大豆、西红柿进行检测并用实时荧光PCR方法进行验证。结果：在79份抽检样品中，共检出含有转基因成分的样品4份，总检出率为5．1％。结论：我市食品中转基因成分存在的情况不容忽视，转基因食品未作任何标识的问题普遍存在，值得管理部门高度重视。应加强对流通市场上食品中转基因成分的监测和转基因食品标识的管理。     

转基因食品DNA提取技术研究

目的：建立从食品中提取DNA的方法。方法：采用CTAB法、酶裂解法、SDS沉淀法3种方法提取转基因相关食品中的：DNA，用核酸蛋白分析仪测定核酸的纯度和浓度、PCR扩增真核生物的18S rDNA基因评价提取核酸的质量。用转基因植物通常含有的CaMV35S启动子作为转基因成分的指示标记。结果：3种方法得到的DNA提取物OD260／OD280比值均接近1．6-1．9，CTAB法获得的DNA量较少，酶提取法所得的DNA提取物的量多，但扩增条带荧光较暗。SDS法能收获较多的DNA，PCR扩增效果好。用SDS法提取的DNA进行35S启动子检测，3份转基因样品均出现强阳性结果，而10份非转基因食品均未产生阳性条带。结论：SDS沉淀法提取DNA，具有经济、简便的特点，所提的DNA量适用于PCR检测。     

Taq Man探针检测转基因大豆含量方法的建立及应用

目的 建立TaqMan探针法检测转基因大豆含量的实时定量PCR方法 并应用于市售转基因食品含量的检测.方法 以0,0.1%、0.5%、2.0%、5.0%的转基因大豆RoundUp Ready<'TM>为标准品(Fluka公司),以特异TaqMan探针序列:5'(FAM)-cccactatccttcgcaagaccct-3'(TAMRA),引物序列F:5'-tgatgtgatatctceactgacg-3',R:5'-tgtatcecttgagceatgttgt-3',用实时荧光定量PCR方法 扩增转基因大豆外源基因CP4EPSPS基因,在实验室建立定量转基因大豆检测的标准曲线,并对市场抽检的豆奶粉、素鸡、热狗肠、黄豆等12种豆类食品及制品进行含量分析.结果 在实验室建立了TaqMan探针实时荧光定量PCR检测转基因大豆含量的方法 ,得到Ct值对转基因食品百分含量对数值的标准曲线方程式为Y=-2.8194X 27.1855,相关系数值R<'2>为0.9994.12种市售大豆及制品中五香荼干、豆奶粉、素鸡、热狗肠、薄白叶检测出转基因成分含量分别为2.46%、6.26%、13.95%、0.48%、0.66%,其余7份样品为阴性.结论 建立的TaqMan探针实时荧光定量PCR法能够快速地进行转基因大豆含量的检测,并可应用于市售转基因食品的检测.     

TaqMan探针检测转基因大豆含量方法的建立及应用

目的建立TaqMan探针法检测转基因大豆含量的实时定量PCR方法并应用于市售转基因食品含量的检测。方法以0、0．1％、0．5％、2．0％、5．0％的转基因大豆RoundUp Ready^TM为标准品（Fluka公司），以特异TaqMan探针序列：5’（FAM）-cccactatcettcgcaagaccct-3’（TAMRA），引物序列F：5’-tgatgtgatatctccactgacg-3’，R：5’-tgtatcccttgagceatgttgt-3’，用实时荧光定量PCR方法扩增转基因大豆外源基因CP4EPSPS基因，在实验室建立定量转基因大豆检测的标准曲线，并对市场抽检的豆奶粉、素鸡、热狗肠、黄豆等12种豆类食品及制品进行含量分析。结果在实验室建立了TaqMan探针实时荧光定量PCR检测转基因大豆含量的方法，得到0值对转基因食品百分含量对数值的标准曲线方程式为Y=-2．8194X＋27．1855，相关系数值R^2为0．9994。12种市售大豆及制品中五香茶干、豆奶粉、素鸡、热狗肠、薄白叶检测出转基因成分含量分别为2．46％、6．26％、13．95％、0．48％、0．66％，其余7份样品为阴性。结论建立的TaqMan探针实时荧光定量PCR法能够快速地进行转基因大豆含量的检测，并可应用于市售转基因食品的检测。     

利用聚合酶链式反应方法检测转基因大豆和玉米中的基因修饰物质

采用聚合酶链式反应 (PolymerasechainreactionPCR)来检测转基因大豆 (RR大豆 )和玉米 (Bt 176玉米 )中基因修饰物质 (GeneticallyModifiedOrganismsGMOs)的方法。利用已知GMOs准确含量的转基因大豆和玉米对PCR检测方法进行了探讨 ,检测限可以达到 0 1% ,即 ,可以把食品中含量仅为 0 1%的GMOs检测出来 ,检测为定性检测。检测的步骤包括 :(1)大豆和玉米基因组DNA的提取 ;(2 )对转入的CaMV35s启动子和NOS终止子利用PCR方法进行扩增 ;(3)对食品的house -keeping基因进行PCR扩增 ,以确定我们所检测的食品确实来自于大豆和玉米 ;(4)利用琼脂糖凝胶电泳及XmnI限制性内切酶方法对PCR产物进行描述与确认。RR大豆含有CaMV35s启动子和NOS终止子 ,而Bt 176玉米只含有CaMV35s启动子。由于玉米的淀粉含量较高 ,因此其基因组DNA的提取效果不如大豆 ,最终的电泳图谱也不如大豆的清晰     

Monitoring of MON810 genetically modified maize in foods in Brazil from 2005 to 2007

Regulations for the use and labeling of genetically modified (GM) products and derived ingredients were implemented in Brazil in 2003. In 2008, GM maize line MON810 was approved for commercialization in Brazil; nevertheless, maize Bt11, Bt176 and MON810 were found in Brazilian market products sold in 2000 and 2001. Nested polymerase chain reaction (PCR) method was employed to monitor the presence of MON810 in 81 maize-derived products (maize flour, corn meal, maize flour flakes and polenta) that are sold in Brazilian markets from 2005 to 2007, after the implementation of genetically modified organism (GMO) labeling regulation. The sensitivity of nested PCR for MON810 detection was 0.1%. CTAB protocol was suitable for the extraction of amplifiable maize DNA from maize flour, corn meal, maize flour flakes, and polenta. MON810 GM maize line was not detected by specific nested PCR in 81 samples of maize-derived food sold in Brazil from 2005 to 2007. This paper reports data concerning the detection of GM maize after publishing Brazilian regulations that stipulate directions for using and labeling of foods and ingredients containing GMOs.     

Survey of compliance with labeling legislation in food containing GMOs in Brazil

Analysis of food products consisting of, or produced from, genetically modified organisms (GMOs) is required to verify compliance with labeling legislation and to detect any possible unauthorized transgenic crops. With these goals, 240 samples of soy-derived foodstuffs and 25 samples of maize-derived foodstuffs were analyzed from 2004 to 2007. All samples positive for Roundup Ready^® soybean were quantitatively examined using the TaqMan^® GMO 35S Soy kit. In food containing soy, 68 (28.3%) were shown to contain GM soy, whereas in food containing maize, neither Bt 176 nor MON 810 maize were found. Quantitative analysis revealed GMO contents ranging from 0.05 to 1% in 43 (63.2%) samples, and more than 1% in 25 (36.8%) samples. The absolute and relative limits of detection (LODs) were approximately 10 copies and 0.0125%, respectively, and the absolute and relative limits of quantification (LOQs) were approximately 40 copies and 0.05%, respectively, suggesting sufficient sensitivity to quantify genetically modified (GM) materials below and above the legal threshold of 1%. The presence of GM material in these samples was not indicated on their labels, indicating that none of these food products had been appropriately labeled. These results clearly demonstrate the need for a monitoring program of food products by the Brazilian regulatory authorities.     

Subacute effects of transgenic crylab bacillus thuringiensis corn litter on the isopods Trachelipus rathkii and armadillidium nasatum

Laboratory studies were conducted to investigate the subacute effects of transgenic Cry1Ab corn leaf material containing Bacillus thuringiensis (Bt) protein on the terrestrial isopods Trachelipus rathkii and Armadillidium nasatum. Survival and growth were measured for eight weeks in isopods fed leaf material of two Bt11 corn varieties, two Monsanto 810 (Mon810) corn varieties, and the isolines of each. Total lipid and protein content of the organisms was measured to examine effects on energetic reserves. Armadillidium nasatum individuals in all treatments responded similarly. For T. rathkii, no statistically significant effect of Bt was observed, but statistical differences were observed in growth between hybrids. Protein and sugar content of the food were found to be correlated with the differences in growth for T. rathkii. Total protein content was higher in T. rathkii and A. nasatum fed material with higher protein and sugar content. A trend toward less growth in T. rathkii on Bt corn varieties versus their isolines triggered a concentration-response assay with purified Cry1Ab protein. No adverse effects of purified Bt protein were observed. These results indicate that little hazard to T. rathkii and A. nasatum from Bt corn leaf material from these hybrids exists. However, nutritional differences in corn hybrids contributed to differences in isopod growth.     

Monitoring of GMO in Brazilian processed meat and soy-based products from 2007 to 2008

Roundup Ready? (RR) soybean is the first genetically modified organism (GMO) approved in Brazil. In this country, the labeling of foodstuffs containing GMOs is mandatory if the GMO content exceeds 1% (10 g kg^?1). In order to monitor and quantify the presence of RR soybean in soy-based and processed meat products on the Brazilian food market, DNA was extracted from 59 food samples and analyzed by polymerase chain reaction (PCR) to amplify the soybean lectin gene, and by nested PCR to amplify the recombinant DNA present in RR soybean. Positive samples for the presence of RR soybean were subjected to a real-time quantification of GMO using a duplex real-time PCR. The results showed that out of 59 samples, 54 were positive for lectin gene and six were positive for RR soybean, 5 samples contained less than10 g kg^?1 GMO and one sample contained more than 10 g kg^?1 GMO.     

Detection of genetically modified DNA sequences in milk from the Italian market

The possible transfer and accumulation of novel DNA and/or proteins in food for human consumption derived from animals receiving genetically modified (GM) feed is at present the object of scientific dispute. A number of studies failed to identify GM DNA in milk, meat, or eggs derived from livestock receiving GM feed ingredients. The present study was performed in order to: (i) develop a valid protocol by PCR and multicomponent analysis for the detection of specific DNA sequences in milk, focused on GM maize and GM soybean; (ii) assess the stability of transgenic DNA after pasteurization treatment and (iii) determine the presence of GM DNA sequences in milk samples collected from the Italian market. Results from the screening of 60 samples of 12 different milk brands demonstrated the presence of GM maize sequences in 15 (25%) and of GM soybean sequences in 7 samples (11.7%). Our screening methodology shows a very high sensitivity and the use of an automatic identification of the amplified products increases its specificity and reliability. Moreover, we demonstrated that the pasteurization process is not able to degrade the DNA sequences in spiked milk samples. The detection of GM DNA in milk can be interpreted as an indicator of fecal or airborne contamination, respectively, with feed DNA or feed particles, although an alternative source of contamination, possibly recognizable in the natural environment can be suggested. Further studies, performed on a larger number of milk samples, are needed to understand the likely source of contamination of milk collected from the Italian market.