Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR
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Food Anal. Methods https://doi.org/10.1007/s12161-017-1097-6 In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR Annalisa Paternò1 & Daniela Verginelli1 & Pamela Bonini1 & Marisa Misto1 & Cinzia Quarchioni1 & Enrico Dainese2 & Stefania Peddis 1 & Cristiana Fusco 1 & Daniela Vinciguerra1 & Ugo Marchesi1 Received: 24 February 2017 /Accepted: 8 November 2017 # The Author(s) 2017. This article is an open access publication Abstract Common wheat is one of the most important staple Keywords Validation . Wheat . Real-time PCR . Droplet food crops worldwide. However, unlike other important staple digital PCR . Taxon-specific . Endogenous reference gene . crops such as maize or soybean, genetically modified (GM) Genetically modified organism . GMO wheat is not yet present in the global food market. Nonetheless, in the recent past, the adventitious presence of GM glyphosate- tolerant volunteers was reported in open wheat fields in the USA. The European Union Reference Laboratory for GM Introduction Food and Feed (EURL-GMFF) was therefore called to develop a strategy to detect such unauthorised GM wheat in wheat Common wheat (Triticum aestivum) is one of the major food samples by using both taxon-specific and screening tests. crops in the world. Belonging to the Triticum genus, it is one Two candidate common wheat taxon-specific real-time PCR of the most widespread staple cereals in the food industry, and methods were suggested, one targeting ssII-D gene coding for consists of several cold-resistant cultivars allowing its cultiva- starch synthase and the other targeting waxy-D1 gene, coding tion in frigid climate areas too. The genus Triticum includes for granule-bound starch synthase. In the present study, the two very complex polyploid genomes, symbolised by the letters above-mentioned real-time PCR taxon-specific methods were A, B, D and G. Common wheat (T. aestivum) is hexaploid in-house verified and compared, proposing droplet digital PCR AABBDD (1C value = 17.33 pg; Bennett and Smith 1976) (ddPCR) as a new tool for supporting the application of the and Triticum durum is tetraploid AABB. European Network of GMO Laboratories (ENGL) established Betting on its potential impact in the food sector, the public method performance criteria. Preliminary performance data of research biotech industry has been striving for decades to put waxy-D1 and ssII-D methods in ddPCR format are shown too genetically modified (GM) wheat on the market by trying to to give a contribution to the bridging process from the consol- incorporate desired traits such as herbicide tolerance, disease idated to the emerging quantitative PCR methodology. resistance and improvements in the quality of this crop (Leckband and Lörz 1998; Anand et al. 2003;Huetal. Electronic supplementary material The online version of this article 2003;Abouseadaaetal.2015;Shinetal.2008). Within this (https://doi.org/10.1007/s12161-017-1097-6) contains supplementary line of research, in the 90s, Monsanto, one of the major material, which is available to authorized users. agrobiotech companies, launched a programme for the devel- opment of genetically modified common wheat tolerant to * Ugo Marchesi glyphosate herbicide, and some promising GM Bevents^ (such [email protected] asMON71800)wereauthorisedintheUSAtobetestedin field trials. However, after completion of the experimental 1 National Reference Laboratory for GM Food and Feed, Biotechnology Unit, Istituto Zooprofilattico Sperimentale del Lazio e phase, no GM wheat has ever been marketed. Nevertheless, Toscana, M. Aleandri via Appia Nuova 1411, 00178 Rome, Italy in 2013, the presence in Oregon (USA) of unexpected glyph- 2 Faculty of Bioscience, Technology for Food Agriculture and osate tolerant wheat was noticed and then confirmed and re- Environment, University of Teramo, Piazza A. Moro 45, ported by the United States Department of Agriculture 64100 Teramo, Italy (USDA). This event was ascribed to MON71800 volunteers, Food Anal. Methods most likely originating from the GM wheat field trials (United Digital PCR platforms currently available are essentially States Department of Agriculture (USDA) 2015). Moreover, classified by the way partitioning of reactions is obtained, ei- in 2016, a similar situation was found in Washington state due ther making use of microfluidic chambers or running individ- to the adventitious presence of a MON71800 sister GM event ual reactions in droplets generated in a water-oil emulsion. The named MON71700 (United States Department of number of references to dPCR and its possible application in Agriculture–USDA news 2016). As this GM crops are not the field of GMO quantification have dramatically increased in authorised in the EU, in both cases, the US Animal and recent years (Burns et al. 2010; Corbisier et al. 2010;Morisset Plant Health Inspection Service (APHIS) informed the et al. 2013;DemekeT.etal.2014; Dobnik et al. 2015;Koppel European Commission and the member states of the and Bucher 2015;Dobniketal.2016;Félix-Urquídezetal. European Union of the event and the European Commission 2015;Gerdesetal.2016; Zhu et al. 2016). urged the member states to start testing consignments of com- In the lack of an event-specific method for the identifi- mon wheat at import from the USA for the presence of cation of the above-mentioned unauthorised GM wheat unauthorised GM wheat. events, the European Union Reference Laboratory for GM The authorisation process for introducing GM products Food and Feed (EURL-GMFF) developed a strategy to de- in the EU market is ruled by Directive 2001/18/EC tect GM wheat in wheat samples by using both taxon- (European Parliament and Council of the European Union specific and screening tests. This strategy was then de- 2001), for the deliberate release into the environment of scribed in a guidance document published on the EURL- genetically modified organisms, and by Regulation (EC) GMFF website (European Union Reference Laboratory for 1829/2003 (European Parliament and Council of the GM Food and Feed 2013a), together with a comprehensive European Union 2003) for GM food and feed. According review and related bioinformatics analysis on all available to this legislation, in addition to the data concerning risk relevant literature on taxon-specific real-time PCR systems assessment, the applicant must provide a method for the for the identification of T. aestivum (European Union detection and quantification of the transformation event un- Reference Laboratory for GM Food and Feed 2013b). The der authorisation. The method has to be validated by the results of the study suggested that the method described by European Union Reference Laboratory for GM Food and Matsuoka et al. (2012), targeting ssII-D gene coding for Feed (EURL-GMFF), established at the Joint Research starch synthase, and the one described by Iida et al. Centre and assisted by the European Network of GMO (2005), targeting waxy-D1 gene, coding for granule-bound Laboratories (ENGL), according to the document starch synthase, may represent good candidates to specifi- BDefinition of minimum performance requirements for an- cally identify common wheat even in complex food sam- alytical methods of GMO testing-MPR^ (European ples. With regard to the latter method, what was reported in Network of GMO Laboratories (ENGL) 2015). the above-mentioned EURL-GMFF guidelines had to be Quantitative methods are required for enforcing EU label- carefully considered, i.e. Baccording to in silico analyses ling and traceability rules for food/feed containing authorised conducted by the EURL GMFF, the primer sequences de- GM material >0.9% with respect to the specific ingredient. scribedinIidaetal.2005 are not correct. The primers The quantitative analysis consists of a relative quantification (Wx012F/Wx012R) and probe (Wx-Taq 1) sequences re- of the GMO event to the plant species itself (taxon reference ported by the same authors in the following article: BImai system). In this context, the selection of the species-specific et al. (2012), Food Hyg. Saf. Sci. 204 Vol. 53 203–210 and reference system is crucial for the reliability of the quantitative the patent EP2180051^ are recommended as reference. analytical results obtained. In 2016, EURL-GMFF verified an endpoint real-time Real-time PCR represents the gold standard for GMO anal- PCR method submitted by Monsanto Company, for de- ysis, and the MPR guidelines refer exclusively to this tech- tecting event MON71800 in wheat, but the wheat- nique. Nevertheless, digital PCR (dPCR) represents one of the specific system of choice was designed on T. aestivum more recent and promising quantitative PCR techniques. In acetyl-coenzyme A carboxylase, Acc-1, reference target dPCR, the sample is distributed across a large number of par- (Genebank AF029897.1), although it is unable to discrim- titions containing zero, one, or more copies of target DNA. inate between T. aestivum and T. durum (European Union The absolute number of target DNA is then calculated by Reference Laboratory for GM Food and Feed 2016). taking into consideration the ratio of positive (or negative) Given the importance of T. durum in food matrices, such partitions to total partitions and using Poisson distribution an evident lack of specificity was considered to be a ma- model. According to this model, λ is the average number of jor drawback, and therefore, Acc-1 targeting method was target copies per partition, and it is determined