Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

The import and use of genetically modified organisms (GMOs) is strictly regulated in the European Union. In order to maintain the legislation on GMOs, a genetic element screening is generally applied as a first step to detect authorised as well as unauthorised GMOs. Subsequent identification of GMOs that relate to the detected elements is performed by the application of event-specific detection methods. However, as the diversity of GMOs on the world market is increasing, there is an ongoing need for methods for additional informative screening elements. Genes that are increasingly applied in GMOs are cry3A (including variants mcry3A and eCry3.1Ab) conferring resistance to Bt toxins, and gat, detoxifying glyphosate. Novel TaqMan PCR detection methods for element cry3A and construct gat/T-pinII were developed to support the identification of maize MIR604, 98140, 5307, canola 61061 and 73496, and soybean 356043. Also, other unknown (unauthorised) GMOs containing cry3A and/or gat/T-pinII can potentially be detected. Specificity, efficiency and sensitivity of the methods were evaluated

Applicability of the poultry qPCR method to detect DNA of poultry processed animal protein materials

After the Bovine Spongiform Encephalitis (BSE) crisis most processed animal proteins (PAPs) were banned from use in animal feed. For the foreseen reintroduction of pork PAPs in poultry feed, and poultry PAPs in pork feed and to comply with the species-to-species ban that prohibits cannibalism, a sensitive and specific TaqMan PCR detection method for poultry DNA has been designed and published. This poultry method is able to detect DNA of chicken, turkey, duck and geese in one PCR reaction. PAPs however, are a difficult and variable matrix. Therefore, the usability of the poultry method was investigated on a range of different poultry PAPs. It was shown that the poultry detection method is capable of detecting poultry DNA in eight out of nine different poultry PAPs mixed at a 0.1% level in chicken feed. The method can also detect at least 0.1% poultry PAPs mixed in pork PAPs. These results show that the poultry method fulfils the 0.1% detection limit requirement in the EU legislation.</p

Applicability of the poultry qPCR method to detect DNA of poultry processed animal protein materials

After the Bovine Spongiform Encephalitis (BSE) crisis most processed animal proteins (PAPs) were banned from use in animal feed. For the foreseen reintroduction of pork PAPs in poultry feed, and poultry PAPs in pork feed and to comply with the species-to-species ban that prohibits cannibalism, a sensitive and specific TaqMan PCR detection method for poultry DNA has been designed and published. This poultry method is able to detect DNA of chicken, turkey, duck and geese in one PCR reaction. PAPs however, are a difficult and variable matrix. Therefore, the usability of the poultry method was investigated on a range of different poultry PAPs. It was shown that the poultry detection method is capable of detecting poultry DNA in eight out of nine different poultry PAPs mixed at a 0.1% level in chicken feed. The method can also detect at least 0.1% poultry PAPs mixed in pork PAPs. These results show that the poultry method fulfils the 0.1% detection limit requirement in the EU legislation.</p

Bridging legal requirements and analytical methods : a review of monitoring opportunities of animal proteins in feed

Availability and safety of food ranks among the basic requirements for human beings. The importance of the food producing sector, inclusive of feed manufacturing, demands a high level of regulation and control. This paper will present and discuss the relationships in the triangle of legislation, the background of hazards with a biological nature, and opportunities for monitoring methods, most notable for prion-based diseases as primary issue. The European Union legislation for prevention of prion-based diseases since 2000 is presented and discussed. The definitions and circumscriptions of groups of species will be analysed in the view biological classification and evolutionary relationships. The state of the art of monitoring methods is presented and discussed. Methods based on visual markers (microscopy), DNA-based methods (PCR), protein-based methods (ELISA, mass spectroscopy, proteomics), near infrared oriented methods and combinations thereof are being evaluated. It is argued that the use in legislation of non-homogeneous groups of species in a biological sense will hamper the optimal design of monitoring methods. Proper definitions are considered to act as bridges between legal demands and suitable analytical methods for effective monitoring. Definitions including specified groups of species instead of single species are more effective for monitoring in a range of cases. Besides the desire of precise circumscription of animal groups targeted by legislation, processed products need well defined definitions as well. Most notable examples are blood versus blood products, and hydrolysis of several types of material. The WISE principle for harmonising the design of legislation and of analytical methods is discussed. This principle includes the elements Witful (reasonable legal principles), Indicative (clear limits between prohibition and authorisation), Societal demands (public health, environment, economy), and Enforceable (presence of suited monitoring methods) in order to promote a balanced effort for reaching the desired level of safety in the food production chain

Data on screening and identification of genetically modified papaya in food supplements

This article contains data related to the research article entitled “A case study to determine the geographical origin of unknown GM papaya in routine food sample analysis, followed by identification of papaya events 16-0-1 and 18-2-4” (Prins et al., 2016) [1]. Quantitative real-time PCR (qPCR) with targets that are putatively present in genetically modified (GM) papaya was used as a first screening to narrow down the vast array of candidates. The combination of elements P-nos and nptII was further confirmed by amplification and subsequent sequencing of the P-nos/nptII construct. Next, presence of the candidate GM papayas 16-0-1 and 18-2-4 were investigated by amplification and sequencing of event-spanning regions on the left and right border. This data article reports the Cq values for GM elements, the nucleotide sequence of the P-nos/nptII construct and the presence of GM papaya events 18-2-4 and/or 16-0-1 in five samples that were randomly sampled to be analysed in the framework of the official Dutch GMO monitoring program for food

Molecular Characterization and Event-Specific Real-Time PCR Detection of Two Dissimilar Groups of Genetically Modified Petunia (Petunia x hybrida) Sold on the Market

Petunia plants with unusual orange flowers were noticed on the European market and confirmed to be genetically modified (GM) by the Finnish authorities in spring 2017. Later in 2017, inspections and controls performed by several official laboratories of national competent authorities in the European Union detected several GM petunia varieties with orange flowers, but also another group of unusually colored flowers. In the latter group, a so far undetected gene coding for a flavonoid 3’5’ hydroxylase (F3’5’H) responsible for the purple color was identified by German and Dutch authorities, suggesting that the petunias found on the markets contain different genetic constructs. Here, a strategy is described for the identification of GM petunia varieties. It is based on an initial GMO screening for known elements using (real-time) PCR and subsequent identification of the insertion sites by a gene walking-like approach called ALF (amplification of linearly-enriched fragments) in combination with Sanger and MinION sequencing. The results indicate that the positively identified GM petunias can be traced back to two dissimilar GM events used for breeding of the different varieties. The test results also confirm that the transgenic petunia event RL01-17 used in the first German field trial in 1991 is not the origin of the GM petunias sold on the market. On basis of the obtained sequence data, event-specific real-time PCR confirmatory methods were developed and validated. These methods are applicable for the rapid detection and identification of GM petunias in routine analysis. In addition, a decision support system was developed for revealing the most likely origin of the GM petunia