Detection of Genetically Engineered Plants by Multiplex PCR Technology
- Details
- Datukishvili N., Kutateladze, T., Gabriadze, I., Vishnepolsky, B., Karseladze, M.
14 Gotua St., Tbilisi 0160, Georgia. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.
Introduction. Developments in plant genetic engineering have resulted in the growth of a vast number of transgenic crops. During the last decades the acreage and diversity of genetically modified plants have been constantly increased worldwide thus causing growing demand for monitoring of genetically modified organisms (GMO) in each country. One of the most important problems of GMO monitoring is to control continuously rising amount of transgenic varieties. There is an urgent need for fast, cheap and reliable GMO detection methods to meet challenges in detection hundreds of GMOs. The aim of this study was to develop and optimize new multiplex PCR-based technologies for time-saving and cost-effective screening of genetically engineered plants.
Methods. The most widely spread GM plants such as soybean and maize were selected as objects for the study and certified reference materials consisting of Roundup Ready soybean (RRS), maize Bt-176 and maize MON 810 were analysed. DNeasy plant mini kit (Qiagen) was applied for genomic DNA extraction. The spectrophotometer and agarose gel electrophoresis was used for the assessment of DNA quantity, purity and integrity. The plant specific, species-specific and GMO-specific DNA sequences were identified and several sets of oligonucleotide primers were designed for them using bioinformatic tools. The DNAs were analysed by plant-specific, species-specific and GMO-specific uniplex PCR. Different combinations of the primers were verified for their suitability for multiplex PCR. The multiplex PCR products were evaluated by agarose gel electrophoresis. The PCR methods were optimized and validated using soybean and maize GMO standards sets for 0, 0.1%, 0.5%, 1%, 2%, 5% GMO.
Results and Discussion. Evaluation of genomic DNAs by spectrophotometer and agarose gel electrophoresis revealed high purity, integrity and sufficient amount of DNA samples. The analysis by plant specific and species-specific uniplex PCR showed existence of the amplifiable template DNA in all samples tested. The GMO-specific uniplex PCR confirmed presence of the appropriate GM material in each DNA analyte. After optimization of the amplification conditions the several multiplex PCR were developed for simultaneous identification of soybean and maize as well as different GMO varieties. The developed pentaplex PCRs enable synchronous identification of the common transgenic elements, such as: cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 5-enolppyruvylshikimate-phosphate synthase (EPSPS) gene, and Cry1Ab delta-endotoxin (Cry1Ab) gene together with soybean-specific lectin gene and maize-specific invertase and zein genes.
Conclusions. The new multiplex PCR methods were developed, optimized and validated for screening of genetically engineered plants. The obtained results exhibited high specificity and sensitivity (at least 0.1%) of the analytical procedure for GMO identification. The developed multiplex PCRs are effective tools for time- and cost-effective detection of transgenic plants and products derived thereof.