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Food and Forensic Molecular Identification: Update and Challenges Review TRENDS in Biotechnology Vol.23 No.7 July 2005 Food and forensic molecular identification: update and challenges Fabrice Teletchea, Celia Maudet and Catherine Ha¨ nni Centre de Ge´ne´ tique Mole´ culaire et Cellulaire, CNRS UMR5534, UCB-Lyon I, 16, Rue Raphael Dubois, 69622 Villeurbanne Cedex, France The need for accurate and reliable methods for animal development of these methods should protect both species identification has steadily increased during past consumers and producers from frauds, and protect animal decades, particularly with the recent food scares and the species from over-exploitation or illegal trafficking. Mol- overall crisis of biodiversity primarily resulting from the ecular authentication or molecular traceability, which is huge ongoing illegal traffic of endangered species. A basedonthePCRamplificationofDNA,hasbeen relatively new biotechnological field, known as species developed in the past ten years and offers promising molecular identification, based on the amplification and solutions for these issues. Furthermore, this field will analysis of DNA, offers promising solutions. Indeed, probably experience tremendous development because: despite the fact that retrieval and analysis of DNA in (i) Most DNA methods developed have proved useful on processed products is a real challenge, numerous almost all organic substrates and will certainly technically consistent methods are now available and become the new legal standards for identification. allow the detection of animal species in almost any (ii) More regulations have introduced safety standards organic substrate. However, this field is currently facing into the chain of food production (e.g. European a turning point and should rely more on knowledge Regulations such as the 2000/104/EC, which estab- primarily from three fundamental fields – paleogenetics, lishes that fish products can enter the commercial molecular evolution and systematics. circuit only if the commercial name, method of production and capture area are clearly labelled or the 2002/1774/EC, which bans the intra-species recycling of animal by-products). Introduction (iii) During the past decade, molecular identification tests Recent food scares (e.g. BSE, avian flu, foot-and-mouth have only been developed for a few species but it is disease, etc.), malpractices of some food producers, likely that this number will steadily increase, religious reasons, food allergies and GMOS have tremen- particularly among fish (Box 1). dously reinforced public awareness regarding the compo- Therefore, it is now crucial to reassess the different sition of food products. However, because labels do not molecular methods available for animal species identifi- provide sufficient guarantee about the true contents of a cation, particularly in light of three fundamentals fields: product, it is necessary to identify and/or authenticate the paleogenetics, molecular evolution and systematics. We components of processed food, thus protecting both are convinced that these three fields could improve the consumers and producers from illegal substitutions [1]. potential of analysing DNA in degraded substrates, help In addition, trade of endangered species has contrib- to choose the most appropriate molecular markers and uted to severe depletion of biodiversity. Approximately highlight some common problems encountered in sys- 10–20% of all vertebrates and plant species are at risk of tematics that could result in erroneous identification. extinction over the next few decades (IUCN; http://www. Here we concentrate mainly on species identification and redlist.org and CITES http://www.cites.org). Wildlife and not in the recognition of distinct populations of the same their products represent the third greatest illegal traffic species because the latter question requires different after drugs and arms [2] and one of the most serious concepts at some point. threats to the survival of animal populations is poaching. Each year, millions of endangered animals are illegally killed or captured for private zoo collections, hunt DNA from food and forensic samples trophies, ornamental objects (e.g. elephant ivory [3]), Fresh food products or forensic samples without proces- human consumption (e.g. sea turtles and their eggs [4])or sing are suitable for many types of molecular or protein traditional medicine (e.g. tiger [5,6], rhinoceros [7]). analyses (traditional biochemical approaches based on Food authentication and protection of biodiversity both proteins used either electrophoretic, chromatographic or require reliable and accurate methods for determining, immunological techniques; reviewed in [8]). Unfortu- without ambiguity, the animal species in a wide array nately, because most foodstuffs and forensic samples are of degraded and processed substrates (Table 1). The processed, DNA is usually altered. However, several Corresponding author: Ha¨nni, C. ([email protected]). research fields had already worked with such DNA: Available online 31 May 2005 ancient DNA studies or paleogenetics (studying DNA www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.05.006 360 Review TRENDS in Biotechnology Vol.23 No.7 July 2005 Table 1. Examples of forensic or food substrates for molecular analysis Samples analyzed Species targeted Extraction methods Fragment targeted Fragment size (bp) Refs Food products Canned products Tuna Chloroform, methanol, Cyt b 171 [58] water Canned products Sardine Chelexa, phenol, chloroform, Cyt b 152 [59] isoamyl alcohol Meat-and-bone meal in Beef, sheep, pig, chicken Guanidium thiocyanate tRNALys, ATPase 145–313 [60] compound feeds (GUSCN) Blood or meat meal, pet Ruminant, avian, fish and Dneasy tissue kitb 12s rRNA, tRNAval, 104–290 [61] food, baby food pig 16S rDNA ‘Mortara’ salami Goose Genomic prep kitc Cyt b 350 [62] Goat cheese Beef Dneasy tissue kitb D-loop 413 [63] Foie gras Goose and mule duck Phenol, chloroform, isoamyl 5S rDNA 250–1000 [56] alcohol Caviar Sturgeon species Not indicated Cyt b, 16S, 12S Not indicated [55] Forensic products Dried, salted and unfrozen Whale Not indicated D-loop 155–378 [64] strips of meat Formalin fixed paraffin Human Chelexa, QIAmpb Microsatelittes, 128–250 [65] embded tissues Amelogenin Skin, tanned hide, scales Chinese alligator Phenol/chloroform Cyt b 180 [66] Pills and plasters made with Tiger Chelex1, phenol, chloroform Cyt b 165 [6] tiger’s bone Elephant tusk (Ivory) African elephant QIAamp kitb 12S, cyt b, micro- 70–251 [3] satellites Faeces Tiger Guanidium thiocyanate Cyt b 510 [67] (GUSCN) Muscle, blood, eggs, skin Sea turtles Phenol-chloroform; Dneasyb Cyt b 875–876 [4] Soup, dried fin, cartilage Shark Phenol-chloroform; Dneasyb Cyt b 155–188 [68] pills aBioRad (http://www.bio-rad.com). bApplied Biosystems (http://www.appliedbiosystems.com). cAmersham Biosciences (http://www1.amershambiosciences.com). from fossil bones or ancient organic remains), human genetic code and the presence of many non-coding regions, forensics (studying DNA from hairs, saliva, blood etc. from DNA provides much more information than proteins crime scenes), non-invasive ecological studies (studying do. However, in processed products, DNA is altered and DNA from animal faeces or hairs found in the field) and displays several particular features that must be taken more recently, food authentication. Taken together, these into account. fields have demonstrated that, (i) despite being altered, DNA is more resistant and thermostable than proteins Substrates, DNA quality and contaminations are and it is still possible to PCR amplify small DNA Short DNA fragments. First, during production processes, fragments (with sufficient information to allow identifi- food products might be subject to thermal treatments cation) and (ii) DNA could potentially be retrieved from (cooking, pasteurization, etc.), high pressure, pH modifi- any substrate because it is present in almost all cells of an cations, irradiation, drying and so on. For example, many organism. In addition, molecular evolution and phyloge- food products are heated up to 1008C for 10–60 min and netics have shown that, because of the degeneracy of the are exposed to a pH!4. Consequently, molecular Box 1. Review of species identified To evaluate the species for which at least one method is currently O1200 species are fished (http://www.fishbase.org/search.cfm)and available, we attempted to collate comprehensively, but not exhaus- w220 are farmed (both numbers include shellfish) [72]. Nevertheless, tively, all studies published in the past decade. It seemed that almost as with the two previous groups, we observed a strong bias towards all of these studies (w100 reviewed) had focused on a few species several species, such as tuna (20%), salmon (16%) and sturgeon (6%). belonging to three main groups of vertebrates: mammals (36%), By contrast, the most valuable fish have hardly been studied, such as actinopterygian fishes (34%) and birds (20%). sardines [73,59] and cod [74], which are the first (22 472 563 metric † Within mammals, more than one-third (41%) of the studies only tons in 2002) and second (8 392 479 metric tons in 2002) most dealt with one or all of the same four livestock species (cattle, pig, important groups ‘harvested’ worldwide (http://www.fao.org), sheep and goat). The remaining mammal species studied are usually respectively. endangered ones, such as seven species of animals for bushmeat [69], † Finally, we found several studies (10%) that focused on other rhinoceroses [7] or tigers [5].
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