Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

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.

Analysis of grape Vitis vinifera L. DNA in must mixtures and experimental mixed wines using microsatellite markers.

Because wine quality highly relies on the varietal composition of the must, the development of methods allowing the authentication of varieties in musts and wines would be of great value as a guarantee of quality. Microsatellite markers have already been applied to the authentication of grape juices (Faria, M. A.; Magalhães, R.; Ferreira, M. A.; Meredith, C. P.; Ferreira Monteiro, F. J. Agric. Food Chem. 2000, 48, 1096-1100) and to the analysis of experimental wines (Siret, R.; Boursiquot, J. M.; Merle, M. H.; Cabanis, J. C.; This, P. J. Agric Food Chem. 2000, 48, 5035-5040). In the present paper, we accessed the usefulness of this technology for the analysis of must and wine mixtures. The detection limit of DNA mixtures was first estimated on DNA extracted from leaves: 4% of a foreign DNA can be detected. Analysis of must and wine mixtures (Chardonnay B/Clairette B and Syrah N/Grenache N) was performed on experimental fermentations. DNA was extracted along the fermentation process and analyzed using five microsatellite loci. The 70:30 (v/v) mixtures were successfully analyzed until the end of the fermentation. The applications of these results to commercial purposes are discussed.

The international standard ISO/TS 21872-1 to study the occurence of total and pathogenic Vibrio parahaemolyticus and Vibrio cholerae in seafood: ITS improvement by use of a chromogenic medium and PCR.

During two surveys conducted in 2008 and 2009, the culture method described in the international standard ISO/TS 21872-1 was applied to the detection of Vibrio parahaemolyticus and Vibrio cholerae in 112 living bivalve mollusc samples, with a chromogenic medium used in addition to the TCBS agar, as second selective isolation medium and for enumeration of V. parahaemolyticus and V. cholerae by surface inoculation. A PCR method for detection of these 2 Vibrio species and the hemolysin genes tdh and trh, was applied in parallel. In 2009, the survey was extended to finfish fillets and crustaceans. PCR was also used for species confirmation of characteristic colonies. The identity of the PCR products, specifically targeting V. parahaemolyticus, was checked by sequencing. Occurrence of V. parahaemolyticus and V. cholerae isolates in living bivalve molluscs ranged from 30.4% to 32.6% and from 1.4% to 4.7% respectively. In frozen crustaceans (2009 survey) V. parahaemolyticus and V. cholerae isolates were respectively found in 45% and 10% of the samples. No V. parahaemolyticus or V. cholerae was detected in frozen fish fillets, neither by the ISO method nor by PCR. In 2009, enteropathogenic V. parahaemolyticus (trh+) was isolated from 4 out of 43 oyster samples while the trh gene was present in V. alginolyticus strains and in samples where V. parahaemolyticus was not detected (9 over 112 samples). The ISO method failed to isolate V. parahaemolyticus in 44% to 53% of the living bivalve molluscs where PCR detected the toxR gene specific of V. parahaemolyticus (Vp-toxR). Our results highlighted the need for a revision of the ISO/TS 21872-1 standard, at least, for analysis of living bivalve molluscs, and confirmed the increasing concern of enteropathogenic V. parahaemolyticus in French bivalve molluscs. Enrichment at 41.5°C was questioned and some reliable solutions for the improvement of the ISO/TS 21872-1 method, such as the PCR method for screening of positive samples and confirmation of colonies, were pointed out.

Detection of total and pathogenic Vibrio parahaemolyticus in shellfish: comparison of PCR protocols using pR72H or toxR targets with a culture method.

PCR protocols directly applied to enrichment broth cultures were compared with a culture method based on the ISO reference for detection of Vibrio parahaemolyticus in 57 natural bivalve mollusc samples. Comparisons were made on different primer pairs specifically targeting the V. parahaemolyticus-specific toxR gene (Vp-toxR) and pR72H fragment, and also tdh and trh hemolysin genes. The PCR method using these different primer pairs and the culture method were also examined for their limits of detection (LOD). The LODs ranged from 7-24 pg of purified DNA per reaction tube (RT) for primer pair Vp-toxR, but for primer pair pR72H, varied greatly depending on the V. parahaemolyticus strains used (0.7 pg-10.6 ng/RT). The Vp-toxR and pR72H primers allowed the detection of V. parahaemolyticus in 25 and 8 out of the 57 samples, respectively, while only 3 V. parahaemolyticus-positive samples were obtained by the culture method. The effective presence of V. parahaemolyticus in the Vp-toxR-positive samples was confirmed by sequencing the PCR products. The trh and Vp-toxR genes were simultaneously detected in 14% of the samples, which were thus considered as presumptively contaminated with pathogenic V. parahaemolyticus. These results emphasize the need for an efficient survey of both the total and pathogenic V. parahaemolyticus present in seafood in France. The PCR protocol targeting Vp-toxR followed by tdh and trh genes is an efficient and reliable method for the detection of total and presumptively pathogenic V. parahaemolyticus in bivalve molluscs.