Detection method for genetically modified papaya using duplex PCR

A simple and rapid method for the identification of genetically modified (GM) papaya, derived from Line 55-1, was developed by modifying the Japanese official PCR method. Genomic DNA was directly extracted from the fresh fruit without the lyophilization step, using a commercial silica-based kit. To develop a duplex PCR method which simultaneously detects the GM papaya-specific gene and the intrinsic papain gene, the papain 2-5'/3' (amplicon size; 184 bp) primer pair for the detection of the papain gene was newly designed within the region of the products (211 bp) amplified using the papain 1-5'/-3' primer pair adopted in the Japanese official PCR method. To detect the GM papaya-specific gene, the primer pair Nos C-5'/CaM N-3' described in the Japanese official method was used. The DNA sequences of the GM papaya gene and the intrinsic papain gene were co-amplified using the PCR method in a single tube. The developed duplex PCR method allows the simultaneous detection of the products by means of agarose gel electrophoresis or microchip electrophoresis. The proposed method for GM papaya identification is simple and rapid.     

DNA extraction method using a silica-base resin type kit for the detection of genetically modified papaya

Genetically modified (GM) papaya has not yet been approved for importation into, or cultivation in the European Union (EU) and Japan. A DNA extraction method using the Qiagen DNeasy Plant Mini Kit (PM method) and a method using a buffer containing cetyltrimethyl ammonium bromide (CTAB method) have been adopted as the official Japanese methods for detecting GM foods. However, the amounts of DNA extracted from papaya by these methods are very low. Therefore, we investigated an extraction method to obtain a high yield of DNA from raw or freeze-dried fresh papaya using the Promega Wizard DNA Clean-Up Resin System (WCR). The incubation for the extraction was carried out at 58 degrees C without proteinase K for 15 min. The extract was applied to a mini-column, then the column was washed with 80% isopropyl alcohol, and genomic DNA adsorbed on the column was eluted with TE buffer. The WCR method gave a higher yield of genomic DNA, and was simpler and faster than the PM method or CTAB method. In addition, it could be used to extract genomic DNA from fresh papaya at various stages of ripeness. Based on these results, we propose that the present method using WCR is the most practical and useful way to extract genomic DNA for the purpose of detecting GM papaya.     

Development of a rapid detection method for genetically modified rice using the ultra-fast PCR system

Food Science and Biotechnology - Genetically modified (GM) rice varieties containing traits such as tolerance to abiotic stress and resistance against pests and diseases continue to be developed....     

A detection method for recombinant DNA from genetically modified maize CBH351

A method using polymerase chain reaction (PCR) was designed for the detection of genetically modified maize CBH351, which has not authorized as safe for use in foods and feeds in Japan yet. We analyzed a recombinant DNA (r-DNA) sequence introduced into CBH351 maize and designed specific primer pairs to amplify a segment including part of the r-DNA. The PCR products obtained by using the designed primer pairs are specific for CBH351 and should prevent false positive results caused by other maizes and other main cereal crops. The r-DNA introduced into CBH351 could be detected from maize samples containing 0.05-0.1% CBH351 maize. This sensitivity is theoretically equivalent to a level of several genome copies and so this technique is a very efficient means to detect CBH351 maize.     

Detection of recombinant DNA from genetically modified papaya

A method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) papaya (55-1 line), of which the mandatory safety assessment has not been finished in Japan because of insufficient data. The papaya intrinsic papain gene was used as an internal control. The results of PCR amplification of the papain gene segment indicated that a commercial silica membrane type kit (QIAGEN DNeasy plant mini) was useful for extraction of DNA from papaya fruit, but not for extraction from canned papaya fruit. On the other hand, a commercial ion-exchange type kit (QIAGEN Genomic-tip) provided enough purified DNA for PCR from canned papaya fruit. Compared with the parental line and other commercial non-GM papayas, the DNA from GM papaya fruit provided specific amplification bands in PCR with five primer pairs (Nos. 2-6) including beta-glucuronidase and neomycin phosphotransferase II gene-specific ones. On the other hand, the primer pairs recognizing these genes showed false-positive results when we used DNAs from canned papaya. Therefore, we recommend that the primer pairs (Nos. 5 and 6) recognizing the sequences derived from two different species of organism should be used in order to detect specifically the GM papaya in canned fruits.     

A detection method of CryIAc protein for identifying genetically modified rice using the lateral flow strip assay

We examined the lateral flow strip assay for identifying unauthorized genetically modified (GM) rice. The GM rice expresses the Bacillus thuringiensis (Bt) toxin, CryIAc protein, which confers tolerance to insects. The recombinant CryIAc protein was prepared from the inclusion bodies of an E. coli. strain into which the CryIAc gene had been inserted, using gel filtration chromatography. The lateral flow strip assay for the identification of GM cotton which also expresses CryIAc protein, was applied to unpolished rice and polished rice spiked with recombinant CryIAc protein. The spiked recombinant CryIAc protein was clearly detected at the level of 0.012 microg/g in both the unpolished and polished rice. After loading of the extract on the strip, a 60 -minute stand time is necessary to clearly detect CryIAc protein. The detection limit was approximately 12 ng CryIAc protein per gram of rice. These results suggest that the lateral flow strip assay for GM cotton can be used to detect CryIAc protein expressed in GM rice.     

Application of a qualitative and quantitative real-time polymerase chain reaction method for detecting genetically modified papaya line 55-1 in papaya products

Genetically modified (GM) papaya (Carica papaya L.) line 55-1 (55-1), which is resistant to papaya ringspot virus infection, has been marketed internationally. Many countries have mandatory labeling regulations for GM foods, and there is a need for specific methods for detecting 55-1. Here, an event- and construct-specific real-time polymerase chain reaction (PCR) method was developed for detecting 55-1 in papaya products. Quantitative detection was possible for fresh papaya fruit up to dilutions of 0.001% and 0.01% (weight per weight [w/w]) for homozygous SunUp and heterozygous Rainbow cultivars, respectively, in non-GM papaya. The limit of detection and quantification was as low as 250 copies of the haploid genome according to a standard reference plasmid. The method was applicable to qualitative detection of 55-1 in eight types of processed products (canned papaya, pickled papaya, dried fruit, papaya-leaf tea, jam, puree, juice, and frozen dessert) containing papaya as a main ingredient.     

A simple DNA extraction method suitable for PCR detection of genetically modified maize

BACKGROUND: The polymerase chain reaction (PCR) is a powerful tool that is being increasingly used for detection of transgenic DNA. PCR requires only a minute quantity of template, but sensitive and accurate testing requires DNA of sufficient purity and free from inhibitors such as plant polysaccharides. Several standard protocols are available for this purpose, but they usually involve several steps, imply destruction of the maize kernel, or are time‐consuming. Our aim was to develop a fast and simple extraction method to isolate a raw DNA‐containing solution from maize tissues suitable for use as a template in a PCR‐based detection assay with specific oligonucleotides directed to the identification of event MON810. RESULTS: The NaOH‐based DNA extraction method we report here is time‐saving (5 min) and can be used to isolate DNA‐containing solutions from a small maize leaf portion (down to 1 mg) or from a single overnight‐germinated kernel. PCR performed with selected primers yielded reproducible detection of transgenic DNA. CONCLUSION: The main advantages of the procedure are the quick extraction step, the possibility of non‐destructive testing of maize kernels, and the robustness of the PCR‐based detection, a consequence of the selection of MON810‐matching oligonucleotides yielding intense and highly specific amplicons. Copyright © 2007 Society of Chemical Industry     

2012年深圳市市售转基因番木瓜检测

对深圳市场转基因番木瓜进行筛查和品系鉴定,为评估市售转基因番木瓜的食用安全风险奠定基础,为政府监管提供依据。方法 在深圳市场随机抽取转基因番木瓜57份,采用实时荧光PCR法,运用大部分转基因植物共有的CaMV35S启动子和NOS终止子进行转基因成分筛查,对筛查出的阳性样品运用各品系特异性的引物探针进行品系鉴定。结果 57份番木瓜样品中,转基因阳性率为91.2%,其中, GMYK16-0-1品系占96.1%,华农1号品系占3.9%,未检出其他品系转基因番木瓜;超市和农产品批发市场的转基因番木瓜阳性率存在明显差异;所有转基因番木瓜均无转基因相关标识。结论 九成以上市售转基因番木瓜为未经我国农业部批准种植的转基因品系,建议政府相关部门加强对转基因番木瓜的监管。     

微生物酶制剂中转基因微生物的实时荧光PCR检测方法

针对微生物酶制剂中残留转基因微生物的检测问题,根据醇氧化酶-1（AOX1）启动子基因的序列信息设计一对引物及一条MGB探针,建立了转基因微生物的实时荧光PCR筛选检测方法。实验结果表明,该方法灵敏度达到1pg,可以准确判定生产线上不同分离阶段的微生物酶制剂中的转基因微生物残留情况。该方法准确度和灵敏度高,操作方便,可作为微生物酶制剂中转基因微生物分离状况的监测方法,也可为其他转基因微生物检测研究提供借鉴和参考。      

微生物酶制剂中转基因微生物的实时荧光PCR检测方法

针对微生物酶制剂中残留转基因微生物的检测问题,根据醇氧化酶-1(AOX1)启动子基因的序列信息设计一对引物及一条MGB探针,建立了转基因微生物的实时荧光PCR筛选检测方法.实验结果表明,该方法灵敏度达到1pg,可以准确判定生产线上不同分离阶段的微生物酶制剂中的转基因微生物残留情况.该方法准确度和灵敏度高,操作方便,可作为微生物酶制剂中转基因微生物分离状况的监测方法,也可为其他转基因微生物检测研究提供借鉴和参考.     

Limits of a PCR-based detection method for genetically modified soya beans in wheat bread production

 The official PCR-based method for the detection of recombinant DNA from glyphosate-tolerant soya beans (GTS), laid down in the collection of methods according to Sect. 35 of the German Food Law, was investigated for applicability. As a model, wheat bread was produced with a 1% addition of baking aid consisting of 45% GTS flour. DNA extraction of samples drawn at various stages of the production process revealed that during the process a degradation of DNA took place, resulting in fragment sizes in bread of <500 bp. GTS DNA was detectable at all stages, although the content of GTS flour in the dough and bread had dropped to only 0.4%. In 2 out of 15 commercial baking aids, GTS DNA was detected, reflecting the status of the use of GTS in that area. A model was also developed to study the effect on the detection of GTS under conditions when the target gene sequence of one primer is present in food originating from natural contamination or genetically modified organisms other than GTS. It was observed that high concentrations of competing DNA inhibited the PCR. This inhibition was overcome by increasing the concentration of Taq polymerase in the reaction mixture. Received: 30 April 1998 / Revised version: 17 June 1998     

A detection method for recombinant DNA from genetically modified potato (NewLeaf Y potato)

A detection method using the polymerase chain reaction (PCR) was developed to detect genetically modified (GM) potato (NewLeaf Y potato; NL-Y), of which the mandatory assessment has not yet been completed in Japan. The potato sucrose synthase gene was used as an internal control. We designed a primer pair to specifically detect NL-Y without false-positive results in processed potato foods infected with the potato virus Y (PVY). The DNA introduced into NL-Y using the primer pair could be detected from potato powder samples containing 0.05% NL-Y. In addition, we designed primer pairs for recognizing the CryIIIA gene to detect the NewLeaf potato (NL), NewLeaf Plus potato (NL-P) and NL-Y and for recognizing p-FMV in order to detect NL-P and NL-Y. The proposed method was applied to the detection of NL-Y in 26 processed potato foods and NL-Y was not detected in any samples.     

转基因油菜籽PCR检测技术研究

以转基因抗草甘膦油菜籽F4—27和转基因抗草丁膦油菜籽T—21为材料,通过使用特异性引物,对转基因油菜籽常见启动子、终止子、筛选标记基因和转入目的基因等多个外源转基因元件进行采用PCR检测,以期建立一套快速、准确、高效转基因油菜籽筛查鉴定技术。     

食品中转基因成分的PCR快速检测研究

为建立食品中转基因成分的快速检测方法，针对转基因大豆、玉米、油菜的多个相对稳定的转基因元件，包括35S、NOS、EPSPS、Cry1A、NPTⅡ等基因，同时选定植物本身固有的基因：大豆Lectin、玉米IVR、油菜Napin基因作为内源参照指示基因，设计、筛选出9对引物分别组成多重PCR．结合高灵敏度的聚丙烯酰胺凝胶电泳组成快速检测体系对转基因食品进行检测，1～2d能完成整个检测过程。经测试：该检测体系稳定可靠、灵敏准确、特异性好，且操作简便、成本低廉，是一种进行转基因食品检测的良好技术模式。     

Detection method for genetically modified potato using an ultra-fast PCR system

Food Science and Biotechnology - Genetically modified (GM) potatoes having resistance to insects and viral diseases, low reducing sugar contents, and black spots for high quality continue to be...     

A PCR-microarray method for the screening of genetically modified organisms

A new method to screen and to identify genetically modified organisms (GMO) is presented in this paper. It is based on the detection of multiple genetic elements common to GMO by their amplification via PCR followed by direct hybridisation of the amplicons on microarray. The pattern of the elements is then compared to a database of the composition of EU-approved GMO and an identification of the GMO is then proposed. The limit of detection of the method was ≤0.1% GMO content (w/w) expressed as the amount of target DNA present in the template for single unprocessed material. The DNA targets were detected both in reference materials and in mixtures with the same detection limit. The specificity for the detection of the different elements was found to be very good with no cross-reaction even in samples with two GMO present at different concentrations. The paper presents examples of GMO identification and discusses the potential and limitation of such approaches and how they can facilitate the work of private and enforcement detection laboratories.     

Study on positive control for GM papaya (55-1) detection method by GUS (beta-glucuronidase) assay

A suitable positive control was investigated for histochemical assay (GUS-examining method) to detect genetically modified (GM) papaya (55-1), currently undergoing a safety assessment in Japan. Six different kinds of test papers were soaked with beta-glucuronidase solution and examined for GUS activity. The test papers made of nylon and glass fiber turned blue, and were stable for fifteen months at -20 degrees C. They are concluded to be useful as positive controls in the GUS-examining method for inspection of GM papaya (55-1).     

Development and application of a selective detection method for genetically modified soy and soy-derived products.

A method has been developed to distinguish between traditional soy beans and transgenic Roundup Ready soy beans, i.e. the glyphosate ('Roundup') resistant soy bean variety developed by Monsanto Company. Glyphosate resistance results from the incorporation of an Agrobacterium-derived 5-enol-pyruvyl-shikimate-3-phosphatesynthase (EPSPS) gene. The detection method developed is based on a nested Polymerase Chain Reaction (PCR) procedure. Ten femtograms of soy bean DNA can be detected, while, starting from whole soy beans, Roundup Ready DNA can be detected at a level of 1 Roundup Ready soy bean in 5000 non-GM soy beans (0.02% Roundup Ready soy bean). The method has been applied to samples of soy bean, soy-meal pellets and soy bean flour, as well as a number of processed complex products such as infant formula based on soy, tofu, tempeh, soy-based desserts, bakery products and complex meat and meat-replacing products. The results obtained are discussed with respect to practical application of the detection method developed.