蜂蜜中转基因成分的PCR检测

我国是蜂蜜生产大国,棉花是主要蜜源植物之一。近些年来,我国种植的棉花大多为转基因抗虫棉,棉花蜜中是否含有转基因成分,引起国内外广大消费者的密切关注。本文以抗虫棉种植地采取的棉花蜜为原料,采用改良CTAB法,在DNA提取过程中,用PBS缓冲液除去其中大部分的可溶性多糖,并提高CTAB提取缓冲液中NaCl浓度以去除残余多糖,从蜂蜜中成功提取出片段完整、纯度较高的DNA,DNA得率为486ng/mL,OD260/OD280为2.01,能够满足后续PCR定性检测的需求。从以转基因棉花为蜜源的蜂蜜中检测出外源DNA片段,建立了蜂蜜中转基因成分的PCR检测技术。本研究对转基因食品标识制度的完善、转基因食品监管、减少贸易摩擦以及保证消费者权益等具有重要意义。     

转基因植物食品检测技术研究进展

随着转基因植物的迅速发展,转基因植物（GMO）含量大量涌向市场。出于对人类健康的考虑,GMO食品标识制度在世界范围内受到人们普遍关注。不论是对GMO食品贴标签,或是对GMO与非GMO原料的分别输送,GMO原料和食品的检测技术是必不可少的。GMO食品的检测主要有两种方法：一种是以DNA为基础的PCR法,另一种是从蛋白质水平出的ELISA法。比较全面地阐述了这两种检测方法的应用状况,重点介绍了PCR检测法及影响PCR检测方法的因素,作者预测DNA芯片检测法将是未来的发展方向。     

转基因番木瓜PCR 快速检测体系的优化

随着公众对转基因食品安全性的关注, 转基因番木瓜的快速检测技术成为关键问题。优化了番木瓜总DNA的提取方法, 选用木瓜蛋白酶基因(Papain)作为内源参照基因, 建立了转基因番木瓜GM YK 和华农一号的双重PCR定性检测方法。通过对广州市超市和批发市场222 个样品的检验, 并与SN/T 2653-2010 行业标准对比, 证明该方法具有污染少、速度快、成本低的优点。     

理性认识转基因植物食品的安全性

讨论现代生物技术的几个基本概念 ,如转基因生物、基因修饰生物、重组DNA植物以及对转基因植物食品安全性评价框架 ,阐明作者对转基因植物食品安全性的理解。     

现代生物技术在食品检测中的应用

介绍了DNA探针、PCR技术、免疫检测技术在食品微生物及转基因成分检测中的应用。着重阐述了PCR技术的工作原理、应用及其发展前景。同时简要介绍了生物芯片及其在食品检测中的应用前景     

浅谈转基因农作物的食品安全性研究

简要综述了转基因农作物的食品安全性研究,内容包括转基因农作物中外源DNA特征、目的基因产生的蛋白质,以及转基因农作物的非转基因成分如营养成分、抗营养因子和毒性物质等的分析,对认识和了解转基因食品有一定的帮助.     

出入境转基因产品及其分子检测现状与展望

随着转基因产品在全球的迅速推广,包括我国在内的很多国家都建立了转基因标识制度。各检验检疫口岸应转基因产品生产企业、食品制造商、消费者等多方面需要,相继开展了转基因产品的检测工作。准确可靠的转基因产品检测技术是各国检疫检疫单位的共同需求。转基因产品的检测主要有两大类方法,一类是DNA水平上的检测,另一类是蛋白质水平上的检测。多个发达国家也相继成立专门机构或部门,负责转基因产品生物检测技术标准化工作。国际上对转基因产品的检测工作有向委托鉴定方向发展的趋势。我们简要综述了出入境转基因产品及其分子检测现状。     

Chelex-100快速提取用于转基因检测DNA模板的研究

目的:建立从转基因作物中快速提取DNA的方法.方法 :采用Chelex-100法提取抗草甘膦大豆和非转基因大豆、转基因抗虫玉米Bt176和非转基因玉米中的DNA,使用PCR扩增大豆和玉米的内源基因(Lectin,zSSⅡb)及外源特异性序列(CaMV35S,Bt176)评价提取核酸的质量.结果 :Chelex-100法能够快速在1h之内从大豆和玉米中提取DNA,所提取的DNA可以直接用于PCR扩增反应,PCR扩增产物电泳条带清晰,转基因抗草甘膦大豆样品和转基因抗虫玉米Bt176检测均出现强阳性结果.结论 :Chelex-100法提取DNA可以作为转基因检测的模板,该方法具有经济、简便、快速的特点,适合于转基因检测工作.     

基于数字PCR的转基因水稻LL62品系精准定量检测方法建立

为了促进转基因产品标识制度的顺利实施,对未获我国农业部批准的转基因水稻LL62品系进行精准定量检测方法研究,以保障我国进出口转基因农产品的检测监管。基于转基因水稻LL62品系外源插入片段和水稻基因组DNA的3'端旁侧序列设计数字PCR检测体系,建立了精准定量检测方法。结果显示,设计的引物探针反应效率高,基于此建立的数字PCR方法高度特异于转基因水稻LL62品系检测;方法可重复性好,生成微滴数相对标准偏差(RSD)值介于0.60%-11.11%;准确度高,3个混合样品(10%、1%和0.1%)测定值与真实值之间的偏差(bias)分别为0.12、0.09和0.10,相对标准偏差(RSD)介于0.09%-10.31%。建立的转基因水稻LL62品系微滴数字PCR定量检测方法简便快捷、特异性强、重复性好、准确度高,可准确有效地对农产品和食品中转基因水稻LL62成分进行精准定量分析。     

转基因植物食品中的转基因DNA能在人类胃肠道中存活并转移入胃肠道微生物种群和肠壁上皮细胞吗?

近年来,由于转基因或基因改造(GM)生物技术的普遍应用,科学界已注意到了转基因有可能逸离GM植物,而进入其野生近缘物种(Wilkinson等,Science,302,457~459,2003;Anon.,Phil.Trans.Soc.Lond B,358,1775~1913,2003;An-dow,Nat.Biotechnol.21,1453~1454,2003)。但对于目前人类比较普遍地食用GM植物食品后,这些食品中的转基因DNA的真正去向,迄今所知极少。人们常常忧心忡忡地怀着这样那样的疑虑。例如,已知GM食用植物细胞中的大部分转基因DNA,已在食物加工和烹饪过程中被降解,其中的一部分转基因DNA仍然可保持完整。这些残余的转基因…     

Selective delivery of interleukine-1 receptor antagonist to inflamed joint by albumin fusion

Background

There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device.

Results

Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation.

Conclusions

LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.     

Chitosan functionalized magnetic particle-assisted detection of genetically modified soybeans based on polymerase chain reaction and capillary electrophoresis

The high quality of DNA template is one of the key factors to ensure the successful execution of polymerase chain reaction (PCR). Therefore, development of DNA extraction methods is very important. In this work, chitosan modified magnetic particles (MPs) were synthesized and employed for extraction of genomic DNA from genetically modified (GM) soybeans. The extraction protocol used aqueous buffers for DNA binding to and releasing from the surface of the MPs based on the pH inducing the charge switch of amino groups in chitosan modified MPs. The extracted DNA was pure enough (A(260)/A(280)=1.85) to be directly used as templates for PCR amplification. In addition, the PCR products were separated by capillary electrophoresis for screening of GM organisms. The developed DNA extraction method using chitosan modified MPs was capable of preparation of DNA templates, which were PCR inhibitor free and ready for downstream analysis. The whole process for DNA extraction and detection was preferable to conventional methods (phenol-chloroform extraction, PCR, and gel electrophoresis) due to its simplicity and rapidity as well as its avoiding the use of toxic reagents and PCR inhibitors.     

A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR

Rapid nucleic acid-based methods to detect human pathogens in foods are dependent on the reliability of the DNA or RNA extraction method used. Skim milk, non-fat dry milk, Cheddar and Brie cheese, and reconstituted whey powder were seeded with serially diluted (10(0)-10(7) cfu 10 ml(-1)) Escherichia coli O157:H7 and subjected to DNA extraction (i) directly from the food product using a solvent-based procedure and (ii) using a guanidinium isothiocyanate (GITC) procedure after previous bacterial concentration. Both the efficiency of DNA extraction and the overall PCR detection limits were evaluated. In almost all instances, the total DNA yield using the solvent method was greater than that obtained for the concentration method. However, the purity of the DNA obtained after bacterial concentration was significantly better than that obtained after organic extraction alone. PCR detection limits after each DNA recovery method varied with the specific food, ranging from 10(1) to 10(4) cfu ml(-1) for all products except whey powder. DNA yields and subsequent PCR detection limits for reconstituted whey powder were extremely poor, and neither procedural changes nor the addition of PCR enhancement agents were able to improve recovery and/or detection. It is concluded that the efficiency of DNA extraction is an extremely important and frequently overlooked variable impacting the overall detection limits of PCR-based detection strategies.     

Identification of genetically modified vegetable sources in food and feed using hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by the hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in feed and food products. The biochip contained 22 immobilized probes intended for (i) detection of plant DNA; (ii) plant species determination (soybean, maize, potato, rice); (iii) identification of transgenic elements, including 35S CaMV, 35S FMV, rice actine gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, nptII marker genes. The limit of detection was 0.5% of genetically modified (GM) soybean and maize in analyzed samples. Identification of transgenic DNA in food and feed products using either the developed approach or real-time PCR led to virtually identical results. The assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of the GM component based on the identified transgene.     

Inhibitory effects of collagen on the PCR for detection of Clostridium perfringens

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.     

Inhibitory Effects of Collagen on the PCR for Detection of Clostridium perfringens

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.     

A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed

We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5′ ‘HEAD’ region and a 3′ region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1–2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5′ nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.     

Identification of plant-derived genetically modified organisms in food and feed using a hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in food and feed products. The biochip contained 22 immobilized oligonucleotide probes that were intended for (1) detection of plant DNA, (2) determination of plant species (soybean, maize, potato, and rice), and (3) identification of transgenic elements, including sequences of 35S CaMV, 35S FMV, rice actin gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, and nptII marker genes. The limit of detection was 0.5% for genetically modified (GM) soybean and maize in the analyzed samples. The tests on food and feed products using the developed approach and real-time PCR showed full agreement in determination of transgenic DNA in the samples. The proposed assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of GM component based on the identified transgene.     

Comparison of three methods of DNA extraction from cold-smoked salmon and impact of physical treatments

AIMS: To compare three bacterial DNA extraction procedures on cold-smoked salmon (CSS) and assess the impact on their efficiency of two physical treatments of the food matrix, ionizing irradiation and freezing. METHODS AND RESULTS: As molecular methods for bacterial detection have become an important analytical tool, we compared bacterial DNA extraction procedures on CSS. Working with frozen and irradiated CSS, we obtained negative responses from samples known to be highly contaminated. Thus, we decided to study the impact of these two physical treatments on bacterial DNA extraction procedures. The efficiency of bacterial DNA extraction directly from the fish matrix suspension was measured by an rpoB PCR-based reaction. The results demonstrated that the DNeasy tissue extraction kit (Qiagen, Courtaboeuf, France) was the most efficient and reproducible method. We also showed that freezing and ionizing irradiation have a negative impact on DNA extraction. This was found probably not to be due to inhibition as the PCR reaction remained negative after adding BSA to the PCR mix reaction. CONCLUSIONS: The extraction kit was the most efficient method. Physical treatments were shown to hamper bacterial DNA extraction. SIGNIFICANCE AND IMPACT OF THE STUDY: Attention must be paid to molecular bacterial detection on food products subject to freezing or to ionizing irradiation.     

Rapid and economic DNA extraction from a single salmon egg for real-time PCR amplification

Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products.