In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on "Method Verification." (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed.

Biological insecticides are an effective method used in plant protection. One of the most widely used active substances in biological insecticides is Cry1Ab protein, which is toxic for lepidopteran insects. This protein is produced during bacterial sporulation by Bacillus thuringiensis. Other sources of Cry1Ab protein are genetically modified plants (GM) with expression of cry1Ab gene. Cry1Ab protein in both bioinsecticides and GM plants is present in the form of protoxin, which requires activation by enzymatic treatment in the gut of susceptible insects. So far, Cry1Ab mode of action is not fully understood, but there are 3 main concepts describing it. Two of them assume that a toxic protein after binding to receptors in the insect gut penetrates into the cells, causing pore formation in the gut, which leads to the death of the sensitive insect. In the third model Cry1Ab toxic action is a result of toxin-induced chemical processes initiating a cell death pathway. This work describes the structure and mode of action of Cry1Ab protein, present in biological insecticides and genetically modified plants.

The validation of the methods for the detection and quantification of genetically modified organisms (GMOs) is required as a part of genetically modified food and feed authorization in the European Union (EU). Each validated method must meet the minimum performance requirements for GMO testing methods defined at the EU level. This ensures that the National Reference Laboratories (NRLs), which act as the official control laboratories, use reliable, precise, and robust GMO detection and quantification methods. The NRLs demonstrate their competence by obtaining and maintaining accreditation according to the ISO/IEC 17025 standard. The technical requirements of this standard, primarily related to the tests performed in the laboratory, include all factors that determine the required correctness and reliability of each implemented method. In the process of GMO authorization, applicants can submit any method that fulfills the validation criteria. In turn, the validated methods for the detection and quantification of different GM events in the same species often vary regarding the reference gene assay and PCR conditions. This results in the need of multiple PCR analysis of samples with various GM events. Harmonization of the method validation parameters allows for the detection of different GM events in single PCR run, which simplify the routine laboratory work and decrease the costs of performed tests, therefore improving the efficiency of the official control of the EU market. This is particularly important as the number of authorized GMOs in EU for food and feed is continuously growing. In this study, we report successful quantitative real-time PCR method harmonization for 8 of the 10 GM maize events.

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Przed dopuszczeniem na rynek UE produkty zmodyfikowane genetycznie (GM) muszą zostać ocenione pod względem bezpieczeństwa dla zdrowia człowieka i zwierząt a w przypadku uprawy również pod względem bezpieczeństwa dla środowiska. Niezbędnym elementem autoryzacji GMO w UE jako żywność i pasza jest również opracowanie wiarygodnych metod wykrywania i oznaczania ilościowego modyfikacji genetycznych. Metody te umożliwiają identyfikację każdego autoryzowanego GMO i są stosowane przez państwa członkowskie dla potrzeb kontroli rynku. Zgodnie z zaleceniami Komisji Europejskiej do analiz jakościowych i ilościowych GMO wykorzystuje się metody molekularne oparte na technikach PCR i Real Time PCR. Walidacja tych metod, spełniająca określone wymagania, pozwala na uzyskiwanie wiarygodnych, powtarzalnych i porównywalnych wyników pozwalających kontrolować GMO na rynku europejskim.