Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders’ needs.

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

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Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-4875-9) contains supplementary material, which is available to authorized users.

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Nowadays most animal feed products imported into Europe have a GMO (genetically modified organism) label. This means that they contain European Union (EU)-authorized GMOs. For enforcement of these labeling requirements, it is necessary, with the rising number of EU-authorized GMOs, to perform an increasing number of analyses. In addition to this, it is necessary to test products for the potential presence of EU-unauthorized GMOs. Analysis for EU-authorized and -unauthorized GMOs in animal feed has thus become laborious and expensive. Initial screening steps may reduce the number of GMO identification methods that need to be applied, but with the increasing diversity also screening with GMO elements has become more complex. For the present study, the application of an informative detailed 24-element screening and subsequent identification strategy was applied in 50 animal feed samples. Almost all feed samples were labeled as containing GMO-derived materials. The main goal of the study was therefore to investigate if a detailed screening strategy would reduce the number of subsequent identification analyses. An additional goal was to test the samples in this way for the potential presence of EU-unauthorized GMOs. Finally, to test the robustness of the approach, eight of the samples were tested in a concise interlaboratory study. No significant differences were found between the results of the two laboratories.

In 2006 and 2007, field tnals were performed to study the effects of the two IsolatIOn dIstances mdICated by the Dutch CoexIstence Committee, I.e., 25 m between GM (genetICally modIfied) and conventIOnal maIze, and 250 m between GM and dehberately non•GM (e.g., orgamc) maIze, on pollen•medlated gene flow (PMGF) under representative agncultural condItIOns m the Netherlands. Each IsolatIOn dIstance was tested at three dIfferent locatIOns across the Netherlands m both years. For testmg PM G F WIth the 25m IsolatIOn dIstance, GM source fields of 100 m x 100 m (I hal were surrounded by four equally sIzed non• GM receptor fields at a dIstance of 25 m. For testmg PGM F WIth the 250 m IsolatIOn dIstance, I•ha GM source fields were surrounded by four 50 m x 50 m (0.25 hal receptor fields m four dIfferent dIrectIOns at 250m. For the G M source field, the maIze vanety D KC342IYG contammg the M ON 810 event was used WIth both dIstances. A maIze vanety near'lsogemc to the GM vanety was grown m the receptor fields to obtam good flowenng synchromclty between GM and non•GM maIze and thus optImal condItIOns for PG M F. Levels ofthe transgene m gram samples from the receptor fields were measured by a vahdated real•tlme PCR (polymerase cham reactIOn) quantIficatIOn method for the MON8IO event. Analyses showed the followmg levels of gram admIXtUre as a consequence ofPMGF, averaged over 12 fields for each IsolatIOn dIstance tested: at 25 m 0.084% (mdlvldual field averages ranged from 0.009% to 0.296%) m 2006 and 0.080% (0.002% to 0.318%) m 2007, respectively, and at 250 m 0.005% (mdlvldual field averages ranged from 0 to 0.040%) m 2006 and 0.007% (0 to 0.037%) m 2007, respectIvely. Although weather condItIOns clearly dIffered between 2006 and 2007 (a hot and dry summer m 2006 vs. a relatIvely wet one WIth about•average temperatures m 2007), outcrossmg rates dId not dIffer slgmficantly between these years.