Development of a Multiplex Ligation Dependent Probe Amplification (MLPA) Module for Simultaneous Detection of Five Genetically Modified Rapeseed Events — Oak Academic Publishing
Research ArticleOpen AccessGoogle Scholar indexed
Development of a Multiplex Ligation Dependent Probe Amplification (MLPA) Module for Simultaneous Detection of Five Genetically Modified Rapeseed Events
Bavarian Health and Food Safety Authority, Oberschleissheim, Germany
,
Bavarian Health and Food Safety Authority, Oberschleissheim, Germany
,
Bavarian Health and Food Safety Authority, Oberschleissheim, Germany
1 Bavarian Health and Food Safety Authority, Oberschleissheim, Germany
2 Bavarian Health and Food Safety Authority, Oberschleissheim, Germany
3 Bavarian Health and Food Safety Authority, Oberschleissheim, Germany
For the official surveillance of genetically modified crops, efficient and simple detection methods need to be on hand to implement the strict requirements regarding approval, labelling and traceability determined by the European Union. Therefore, a multiplex ligation dependent probe amplification (MLPA) module was developed for simultaneous detection of five genetically modified rapeseed events (MS8, RF3, GT73, Falcon GS40/90 and T45). Probes were designed and concentrations were adapted in order to obtain high sensitivity and specificity. This MLPA module was validated using certified reference materials and its applicability was tested analyzing routine honey, mustard and rapeseed samples. The limit of detection was determined by analyzing a dilution series (n = 16 for each concentration) of respective transgenic DNA. After optimization, the MLPA revealed limits of detection between 10 to 50 copies of the transgene DNA/assay. The method proved to be sensitive and highly reproducible. When analyzing routine samples, results obtained applying the MLPA module were similar compared to real-time PCR.
James, C. (2012) Global Status of Commercialized Biotech/GM Crops: 2012. ISAAA Brief, No. 44, ISAAA, Ithaca, New York.
Engel, K.-H., et al. (2006) Quantification of DNA from Genetically Modified Organisms in Composite and Processed Foods. Trends in Food Science & Technology, 17, 490-497. http://dx.doi.org/10.1016/j.tifs.2006.04.008
Waiblinger, H.-U., et al. (2010) A Practical Approach to Screen for Authorised and Unauthorised Genetically Modified Plants. Analytical and Bioanalytical Chemistry, 396, 2065-2072. http://dx.doi.org/10.1007/s00216-009-3173-2
Elenis, D.S., et al. (2008) Advances in Molecular Techniques for the Detection and Quantification of Genetically Modified Organisms. Analytical and Bioanalytical Chemistry, 392, 347-354. http://dx.doi.org/10.1007/s00216-008-1868-4
Huber, I., et al. (2013) Development and Validation of Duplex, Triplex, and Pentaplex Real-Time PCR Screening Assays for the Detection of Genetically Modified Organisms in Food and Feed. Journal of Agricultural and Food Chemistry, 61, 10293-10301. http://dx.doi.org/10.1021/jf402448y
Waiblinger, H.-U., et al. (2008) Validation and Collaborative Study of a P35S and T-Nos Duplex Real-Time PCR Screening Method to Detect Genetically Modified Organisms in Food Products. European Food Research and Technology, 226, 1221-1228. http://dx.doi.org/10.1007/s00217-007-0748-z
Morisset, D., et al. (2008) Alternative DNA Amplification Methods to PCR and Their Application in GMO Detection: A Review. European Food Research and Technology, 227, 1287-1297.
Schouten, J., et al. (2002) Relative Quantification of 40 Nucleic Acid Sequences by Multiplex Ligation-Dependent Probe Amplification. Nucleic Acids Research, 30, e57. http://dx.doi.org/10.1093/nar/gnf056
Lunkka-Hytonen, M., et al. (2008) G.P.9.04 Development of the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method for Identifying Large Scale Mutations in the Nebulin Gene. Neuromuscular Disorders, 18, 787. http://dx.doi.org/10.1016/j.nmd.2008.06.216
Luber, F., et al. (2012) Apricot DNA as an Indicator for Persipan: Detection and Quantitation in Marzipan Using Ligation-Dependent Probe Amplification. Journal of Agricultural and Food Chemistry, 60, 5853-5858. http://dx.doi.org/10.1021/jf301202s
Mustorp, S.L., Drømtorp, S.M. and Holck, A.L. (2011) Multiplex, Quantitative, Ligation-Dependent Probe Amplification for Determination of Allergens in Food. Journal of Agricultural and Food Chemistry, 59, 5231-5239. http://dx.doi.org/10.1021/jf200545j
Moreano, F., et al. (2006) Ligation-Dependent Probe Amplification for the Simultaneous Event-Specific Detection and Relative Quantification of DNA from Two Genetically Modified Organisms. European Food Research and Technology, 222, 479-485.
Ehlert, A., et al. (2008) Development of a Modular System for Detection of Genetically Modified Organisms in Food Based on Ligation-Dependent Probe Amplification. European Food Research and Technology, 227, 805-812. http://dx.doi.org/10.1007/s00217-007-0790-x
Guertler, P., et al. (2013) Development of a CTAB Buffer-Based Automated gDNA Extraction Method for the Surveillance of GMO in Seed. European Food Research and Technology, 236, 599-606. http://dx.doi.org/10.1007/s00217-013-1916-y
Guertler, P., et al. (2014) Automated DNA Extraction from Pollen in Honey. Food Chemistry, 149, 302-306. http://dx.doi.org/10.1016/j.foodchem.2013.10.129
Dong, W., et al. (2008) GMDD: A Database of GMO Detection Methods. BMC Bioinformatics, 9, 260. http://dx.doi.org/10.1186/1471-2105-9-260
Zuker, M. (2003) Mfold Web Server for Nucleic Acid Folding and Hybridization Prediction. Nucleic Acids Research, 31, 3406-3415. http://dx.doi.org/10.1093/nar/gkg595
Hess, N., Ulrich, A. and Hoffmann, T. (2002) Insertionsspezifische Nachweisverfahren für transgene Pflanzenlinien unter Anwendung der inversen PCR. Bundesgesundheitsblatt-Gesundheitsforschung-Gesundheitsschutz, 45, 626-633. http://dx.doi.org/10.1007/s00103-002-0444-x
Mazzara, M., et al. (2007) Quantitative PCR Method for Detection of Oilseed Rape Event MS8. European Union Reference Laboratory for GM Food and Feed.
Mazzara, M., et al. (2007) Quantitative PCR Method for Detection of Oilseed Rape Event GT73. European Union Reference Laboratory for GM Food and Feed.
Savini, C., et al. (2007) Quantitative PCR Method for Detection of Oilseed Rape Event RF3. European Union Reference Laboratory for GM Food and Feed.
Demeke, T. and Ratnayaka, I. (2008) Multiplex Qualitative PCR Assay for Identification of Genetically Modified Canola Events and Real-Time Event-Specific PCR Assay for Quantification of the GT73 Canola Event. Food Control, 19, 893-897. http://dx.doi.org/10.1016/j.foodcont.2007.08.020
James, D., et al. (2003) Reliable Detection and Identification of Genetically Modified Maize, Soybean, and Canola by Multiplex PCR Analysis. Journal of Agricultural and Food Chemistry, 51, 5829-5834. http://dx.doi.org/10.1021/jf0341159
Yang, L., et al. (2008) Qualitative and Quantitative Event-Specific PCR Detection Methods for Oxy-235 Canola Based on the 3’ Integration Flanking Sequence. Journal of Agricultural and Food Chemistry, 56, 1804-1809. http://dx.doi.org/10.1021/jf073465i
Yang, L., et al. (2006) Event-Specific Qualitative and Quantitative Polymerase Chain Reaction Analysis for Genetically Modified Canola T45. Journal of Agricultural and Food Chemistry, 54, 9735-9740. http://dx.doi.org/10.1021/jf061918y
Taverniers, I., et al. (2005) Event-Specific Plasmid Standards and Real-Time PCR Methods for Transgenic Bt11, Bt176, and GA21 Maize and Transgenic GT73 Canola. Journal of Agricultural and Food Chemistry, 53, 3041-3052. http://dx.doi.org/10.1021/jf0483467