ABSTRACT
DNA-metabarcoding is becoming more widely used for routine authentication of meat-based food and feed products. Several methods validating species identification methods through amplicon sequencing have already been published. These use a variety of barcodes and analysis workflows, however, no methodical comparison of available algorithms and parameter optimization are published hitherto for meat-based products' authenticity. Additionally, many published methods use very small subsets of the available reference sequences, thereby limiting the potential of the analysis and leading to over-optimistic performance estimates. We here predict and compare the ability of published barcodes to distinguish taxa in the BLAST NT database. We then use a dataset of 79 reference samples, spanning 32 taxa, to benchmark and optimize a metabarcoding analysis workflow for 16S rDNA Illumina sequencing. Furthermore, we provide recommendations as to the parameter choices, sequencing depth, and thresholds that should be used to analyze meat metabarcoding sequencing experiments. The analysis workflow is publicly available, and includes ready-to-use tools for validation and benchmarking.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Polymerase Chain Reaction (PCR)-based screening methods targeting genetic elements commonly used in genetically modified (GM) plants are important tools for the detection of GM materials in food, feed, and seed samples. To expand and harmonize the screening capability of enforcement laboratories, the German Federal Office of Consumer Protection and Food Safety conducted collaborative trials for interlaboratory validation of real-time PCR methods for detection of the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus and a construct containing the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens sp. strain CP4 (ctp2-cp4epsps), respectively. To assess the limit of detection, precision, and accuracy of the methods, laboratories had to analyze two sets of 18 coded genomic DNA samples of events LLRice62 and MS8 with the bar method and NK603 and GT73 with the ctp2-cp4epsps method at analyte levels of 0, 0.02, and 0.1% GM content, respectively. In addition, standard DNAs were provided to the laboratories to generate calibration curves for copy number quantification of the bar and ctp2-cp4epsps target sequences present in the test samples. The study design and the results obtained are discussed with respect to the difficult issue of developing general guidelines and concepts for the collaborative trial validation of qualitative PCR screening methods.
