DNA barcoding as new diagnostic tool to lethal plant poisoning in herbivorous mammals.

Reliable identification of plant species in the digestive tract of a deceased animal often represents the major key to diagnose a lethal intoxication with poisonous plants in veterinary pathology. In many cases, identification of the species is challenging or even impossible because the diagnostic morphological features have been degraded, and because the interpretation of such features requires a considerable expertise in plant anatomy and biodiversity. The use of DNA barcoding markers can support or even replace classical morphological assessment. While these markers have been widely used for plant taxonomy, their forensic application to clarify causes of animal poisoning is novel. In addition, we use specific single-nucleotide polymorphisms as fingerprints. This allows for a clear decision even in cases, where the conventionally used statistical e-values remain ambiguous. In the current work, we explore the feasibility of this strategy in a couple of exemplary cases, either in concert with anatomical diagnostics, or in cases where visual species identification is not possible, or where chemical toxin detection methods are not well established, complex, time consuming and expensive.

Benchmarking and Validation of a Bioinformatics Workflow for Meat Species Identification Using 16S rDNA Metabarcoding.

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.

The Barcoding Table of Animal Species (BaTAnS): a new tool to select appropriate methods for animal species identification using DNA barcoding.

Food and feed products derived from animal materials have a long history of being adulterated. Methods for the identification of animal samples based on DNA barcoding are very potent tools to reveal species substitution. Since numerous DNA barcoding methods are available for different taxa, it is challenging to choose an appropriate and verified method for each sample in question. To overcome this obstacle the working group "Molecular biological methods for plant and animal species differentiation" developed the "Barcoding Table of Animal Species". This working group is established through the German food and feed law and is mandated to validate standard methods, especially for the official food and feed control laboratories in Germany. In this paper, a collection of currently available and verified DNA barcoding methods for the identification of animal species is presented as a Microsoft Excel® file-"The Barcoding Table of Animal Species (BaTAnS)". It consists of several components: The method collection, the results collection and a section for reporting new entries and problems. It is focusing on the validity and applicability of DNA barcoding methods to test food and feed products for correct species declaration. Each method is listed with its reference and is verified by at least two laboratories for their applicability. Since additional information will be available in future, this table will be updated regularly. The BaTAnS is an easy tool that helps to choose the right verified method to identify a certain specimen to taxon, genus or species level in food samples.

Results of an International Interlaboratory Trial to Determine Twelve Allergens Using Real-time PCR- and ELISA-based Assays.

To elucidate the capability of laboratories to determine allergen contents, an international interlaboratory trial was conducted using meat products spiked with 12 allergens. The measurement uncertainty was calculated independent of the applied method simulating realistic situations when comparing analysis certificates from different laboratories. The measurement uncertainty was revealed to be in the best cases +/-100%, in the worst cases quantification exhibited a measurement uncertainty of higher than 200% making quantitative analysis impossible. The measurement uncertainty seemed to depend on the analyte and assays used.

Bite through the tent.

The authors report on a young boy who was bitten into his face by an unknown animal while being asleep in a tent. Given the bite marks and the location of the scene, members of the mustelidae and canidae families were the first "suspects." Deoxyribunucleic acid (DNA) recovered from the tent's wall was analyzed with regard to parts of the mitochondrial 12S ribosomal ribunucleic acid (12S rRNA) and cytochrome b (cytb) genes as well as nuclear short tandem repeats (STRs). Since Sanger sequencing revealed a mixed sequence with a strong human component overlying the nonhuman contributor, an animal screening using a duplex real-time polymerase chain reaction (PCR) with an intercalating dye and melt curve analysis was employed. The results were later confirmed by cloning. The applied commercial canine STR kit verified the animal family (canidae) but did not help in discriminating the species due to cross-species amplification. In the presented case, the real-time PCR assay offered the cheapest and fastest method for animal family determination, which then allowed for an appropriate and sample-saving strategy to characterize the causative animal species.

A practical approach to screen for authorised and unauthorised genetically modified plants.

In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236 x 3006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.