The substitution of more appreciated animal species by animal species of lower commercial value is a common type of meat product adulteration. DNA metabarcoding, the combination of DNA barcoding with next-generation sequencing (NGS), plays an increasing role in food authentication. In the present study, we investigated the applicability of a DNA metabarcoding method for routine analysis of mammalian and poultry species in food and pet food products. We analyzed a total of 104 samples (25 reference samples, 56 food products and 23 pet food products) by DNA metabarcoding and by using a commercial DNA array and/or by real-time PCR. The qualitative and quantitative results obtained by the DNA metabarcoding method were in line with those obtained by PCR. Results from the independent analysis of a subset of seven reference samples in two laboratories demonstrate the robustness and reproducibility of the DNA metabarcoding method. DNA metabarcoding is particularly suitable for detecting unexpected species ignored by targeted methods such as real-time PCR and can also be an attractive alternative with respect to the expenses as indicated by current data from the cost accounting of the AGES laboratory. Our results for the commercial samples show that in addition to food products, DNA metabarcoding is particularly applicable to pet food products, which frequently contain multiple animal species and are also highly prone to adulteration as indicated by the high portion of analyzed pet food products containing undeclared species.

The production of bivalve species has been increasing in the last decades. In spite of strict requirements for species declaration, incorrect labelling of bivalve products has repeatedly been detected. We present a DNA metabarcoding method allowing the identification of bivalve species belonging to the bivalve families Mytilidae (mussels), Pectinidae (scallops), and Ostreidae (oysters) in foodstuffs. The method, developed on Illumina instruments, targets a 150 bp fragment of mitochondrial 16S rDNA. We designed seven primers (three primers for mussel species, two primers for scallop species and a primer pair for oyster species) and combined them in a triplex PCR assay. In each of eleven reference samples, the bivalve species was identified correctly. In ten DNA extract mixtures, not only the main component (97.0–98.0%) but also the minor components (0.5–1.5%) were detected correctly, with only a few exceptions. The DNA metabarcoding method was found to be applicable to complex and processed foodstuffs, allowing the identification of bivalves in, e.g., marinated form, in sauces, in seafood mixes and even in instant noodle seafood. The method is highly suitable for food authentication in routine analysis, in particular in combination with a DNA metabarcoding method for mammalian and poultry species published recently.

Insects have the potential to become an efficient and reliable food source for humans in the future and could contribute to solving problems with the current food chain. Analytical methods to verify the authenticity of foods are essential for consumer acceptance. We present a DNA metabarcoding method that enables the identification and differentiation of insects in food. The method, developed on Illumina platforms, is targeting a 200 bp mitochondrial 16S rDNA fragment, which we found to be suitable for distinguishing more than 1000 insect species. We designed a novel universal primer pair for a singleplex PCR assay. Individual DNA extracts from reference samples, DNA extracts from model foods and food products commercially available were investigated. In all of the samples investigated, the insect species were correctly identified. The developed DNA metabarcoding method has a high potential to identify and differentiate insect DNA in the context of food authentication in routine analysis.

Meat species authentication in food is most commonly based on the detection of genetic variations. Official food control laboratories frequently apply single and multiplex real-time polymerase chain reaction (PCR) assays and/or DNA arrays. However, in the near future, DNA metabarcoding, the generation of PCR products for DNA barcodes, followed by massively parallel sequencing by next generation sequencing (NGS) technologies, could be an attractive alternative. DNA metabarcoding is superior to well-established methodologies since it allows simultaneous identification of a wide variety of species not only in individual foodstuffs but even in complex mixtures. We have recently published a DNA metabarcoding assay for the identification and differentiation of 15 mammalian species and six poultry species. With the aim to harmonize analytical methods for food authentication across EU Member States, the DNA metabarcoding assay has been tested in an interlaboratory ring trial including 15 laboratories. Each laboratory analyzed 16 anonymously labelled samples (eight samples, two subsamples each), comprising six DNA extract mixtures, one DNA extract from a model sausage, and one DNA extract from maize (negative control). Evaluation of data on repeatability, reproducibility, robustness, and measurement uncertainty indicated that the DNA metabarcoding method is applicable for meat species authentication in routine analysis.

Seafood is a valuable commodity with increasing demand, traded for billions of USD each year. The volatility in supply chains and fluctuating prices contribute to the susceptibility of the seafood market to food fraud. Analytical methods are required to identify seafood in processed foods to ensure food authenticity and compliance with European laws. To address this need, we developed and validated a DNA metabarcoding method for the authentication of crustaceans and cephalopods in processed food samples, as both are prone to food fraud, especially in mixed products. A ~200 bp barcode of the mitochondrial 16S rDNA was selected as the marker for identification and sequenced on Illumina platforms. The DNA metabarcoding method utilizes two primer systems, one for the amplification of crustacean DNA and another for cephalopods. The crustacean primer system comprises two forward and two reverse primers, while the cephalopod primer system includes three forward and one reverse primer. DNA extracts from reference materials, model foods, processed foodstuffs, and DNA extract mixtures were investigated. Even species with a close phylogenetic relationship were successfully identified and differentiated in commercial samples, while single species were detected at amounts as low as 0.003% in model foods. However, false-negative results were obtained for certain species in DNA extract mixtures, which are most likely due to degraded or low-quality DNA and can best be prevented by optimized DNA extraction procedures. Our DNA metabarcoding method demonstrates strong potential as a qualitative screening tool in combination with other in-house DNA metabarcoding methods for food authentication in routine analysis.