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Over the last two decades, the number of available genetically modified organisms (GMOs) has been increasing greatly and continuously. Concomitantly with the development of transgenic crops, regulations regarding proper labelling of such products have been introduced, which in turn has forced progress in the molecular methods allowing appropriate detection and identification of such organisms. The main molecular technique that is used for this purpose is polymerase chain reaction (PCR), because it is generally accepted as the most sensitive and specific. There is a variety of PCR-based methods applied for detection of GMO, which can be grouped into four different categories depending on their specificity (Miraglia et al., 2004; Holst-Jensen et al., 2012). The first category comprises screening tests targeting for DNA sequences widely used in the construction of various GM crops. The target sequence includes promoters (e.g., 35S promoter of the cauliflower mosaic virus – CaMV 35S promoter) or terminators (e.g., terminator of the cauliflower mosaic virus – CaMV T-35S and terminator of the nopaline synthase gene – T-NOS), which allow proper expression of the introduced transgene as well as selection markers commonly present in vectors used for genetic modification (nptII – neoABSTRACT. Polymerase chain reaction (PCR) has become the main method for detection and identification of genetically modified organisms (GMOs). Multiplex PCR is a variation of this technique allowing simultaneous amplification of several targets in the same reactions. In this study, it was developed, optimized and performed in-house validation of the multiplex PCR method for detection and identification of genetically modified rape lines (GT73, Ms8, Rf3 and T45) in two types of assays: geneand event-specific. The optimized reactions exhibited high specificity, sensitivity, selectivity and accuracy. The limit of detection (LOD) for the gene-specific reaction was 0.01% for each of the tested lines of rape. For the event-specific reaction, LOD was at the same level for most of the GM rape lines except for T45 (LOD = 0.025%). The optimized and validated assays were employed from 2012 to 2015 in our laboratory to analyse 428 samples of animal feedstuffs containing oilseed rape. GM rape was not detected in the analysed feedstuffs until 2013 (only some traces of GM soyabean were found). Since 2014, the GM rape presence has been confirmed in event-specific reactions in more than one third of analysed samples. Received: 25 August 2016 Revised: 7 April 2017 Accepted: 7 June 2017

Multiplex PCR assays for qualitative detection and identification of the GT73, Ms8, Rf3 and T45 varieties of genetically modified oilseed rape