Interlaboratory Validation of a DNA Metabarcoding Assay for Mammalian and Poultry Species to Detect Food Adulteration

Abstract

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.

A next-generation sequencing approach for the detection of mixed species in canned tuna

“…Several NGS-based or metabarcoding methods also exist in the field of meat analysis ( Ballin et al, 2009 ). A metabarcoding method for the identification of mammalian and poultry species in food ( Dobrovolny et al, 2019 ) has already been tested for routine analysis ( Preckel et al, 2021 ) and has been validated in an interlaboratory ring trial in order to harmonize analytical methods for food authentication ( Dobrovolny et al, 2022 ). Based on the data of this ring trial, the authors suggested a threshold of 0.5 % to reliably assess the presence of a species in a food sample ( Dobrovolny et al, 2022 ).…”

“…Regarding quantification, in meat analysis, the determination of the meat content of species is associated with similar problems, such as the use of mtDNA, processing grade and DNA extractability which impact on the quantitative results. Therefore, results are rather considered as rough estimates for the compositions of mixed species in food products ( Cottenet et al, 2020 , Dobrovolny et al, 2022 , Preckel et al, 2021 ). Factors contributing to a bias of PCR-based methods can result from various sources in the analytical process.…”

“…The results were promising; however, a bias was found primarily with skipjack being overrepresented. The determination of species proportions based on the number of NGS reads is known to be difficult and may result from different factors such as the species in the mixture or the processing degree of the samples ( Dobrovolny et al, 2022 ).…”

Recent Advances in OMICs Technologies and Application for Ensuring Meat Quality, Safety and Authenticity

“…Therefore, meat authentication is an important concern to protect consumers from illegal and unwanted ingredients. Accordingly, three papers dealing with meat authenticity, origin, and detection of meat adulteration using OMICs methods were published [ 15 , 16 , 17 ]. Cai et al [ 15 ] proposed a simple and reliable single-tube septuple PCR assay based on mitochondrial DNA to simultaneously recognize seven meat species from pig, beef, sheep, chicken, turkey, goose, and duck.…”

“…Furthermore, the authors validated the method in terms of sensitivity, specificity, robustness, and low costs for broad application to detect the origin of meat in foodstuffs with suspected adulteration. Another interesting study by Dobrovolny et al [ 16 ] consisted of a collaborative work among 15 laboratories (inter-laboratory ring trial) that aimed to harmonize an analytical method based on DNA metabarcoding assay to detect adulteration from poultry and mammalian species. In this European study, each research team received and analyzed 16 anonymously labeled samples (8 samples, 2 subsamples each) containing six mixtures of DNA extract, one DNA extract from a model sausage, and another from maize, considered in this trial as a negative control.…”