Food Control 21 (2010) 10611065

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Development and application of molecular markers for poultry meat identication in food chain
P. Stamoulis, C. Stamatis, T. Saradou, Z. Mamuris *
Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Street, 41221 Larissa, Greece

a r t i c l e

i n f o

a b s t r a c t
Every year, large quantities of poultry and game meat are consumed. Thus, efcient techniques to identify the meat species origin are required which interest traders, consumers and organizations. In this study, two mitochondrial DNA (mtDNA) genes, Cytochrome b (Cyt b) and 12S ribosomal RNA (12S rRNA) were tested as putative discrimination markers in samples of raw and processed poultry meat (chicken, turkey, duck, goose, pheasant, partridge, woodcock, ostrich, quail and song thrush), applying the PCRRFLP technique with universal primers and ten different restriction enzymes. Digestion of 12S rRNA by AciI successfully distinguished all avian species, producing species-specic patterns. We conclude that the 12S rRNA gene is more informative than Cyt b gene for avian species identication purposes. Moderate process treatment did not prevent the species identication, presenting similar patterns with the raw meat. Finally, this method was considered sufcient to detect mixtures of meat, making it a valuable tool for checking possible adulterations. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 29 May 2009 Received in revised form 22 December 2009 Accepted 31 December 2009

Keywords: Avian species Mitochondrial DNA 12S rRNA

1. Introduction The identication of food species is becoming a very important issue for the assessment of food composition and the provision of proper consumer information. Consumers require clear and accurate information in order to make informed choices about their diet and the foods they buy. Furthermore, in the last few years, the consumption of game meat has gained increasing favor among consumers, who appreciate its texture, avour, the low fat and cholesterol content as well as its lack of anabolic steroids or other drugs (La Neve, Civera, Mucci, & Bottero, 2008). The high commercial value of game meat has sometimes induced fraud, such as mislabelling or selling less valuable meat as meat from more appreciated species (Fajardo, Gonzlez, Calleja, Martn, & Garga, 2006). The increasing demand for transparency in the food industry derives either from socio-religious reasons (e.g. vegetarianism, preference for organic products, absence of pork for Jews and Muslims), health concerns (e.g. absence of peanuts, lactose or gluten for individuals with particular allergies) or economic reasons and has provoked a strong demand for appropriate detection methods that allow identication of different species in meat foods and/or of the different components in processed food. For this purpose, numerous analytical methods have been developed based on protein and DNA analysis. Protein based methods include electrophoretic (Zeri, Labie, & Benard, 1992),
* Corresponding author. Tel.: +30 2410565282; fax: +30 2410565290. E-mail address: zmamur@bio.uth.gr (Z. Mamuris). 0956-7135/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2009.12.027

chromatographic (Amstrong & Leach, 1992) and immunological techniques (Hsieh, Sheu, & Bridgman, 1998; Kangethe, Gathuma, & Landqvist, 1986; Patterson & Jones, 1990). However, these methods are often unsuitable for complex food products as they can not differentiate closely related species in processed materials and are time consuming, inadequate and/or expensive (Calvo, Zaragoza, & Osta, 2001; Koh, Lim, Chua, Chew, & Phang, 1998; Saez, Sanz, & Toldra, 2004). On the other hand, molecular authentication or molecular traceability of meat, which is based on the PCR amplication of DNA, has been developed in the past 15 years (see Asensio, 2007; Dalvit, De Marchi, & Cassandro, 2007, for a review) and offers promising solutions for these issues, being more sensitive even in cases that meat has been cured or autoclaved (Arslan, Ilhak, & Calicioglu, 2006). Approaches for meat species identication are based either on nuclear or mtDNA markers such as AFLP (Alves, Castellanos, Ovilo, Sili, & Rodrguez, 2002; Sasazaki et al., 2003), RAPD (Arslan, Ilhak, & Calicioglu, 2005), DNA sequencing or RFLP (Bartlett & Davidson, 1991; Chikuni, Tabata, Saito, & Monma, 1994; Desjardins & Morais, 1999; Meyer, Hoefelein, Luethy, & Candrian, 1995; Partis et al., 2000), species-specic PCR (Che Man, Aida, Raha, & Son 2007) and multiplex PCR (Asensio, 2008; Dalmasso et al., 2003; Di Pindo, Forte, Conversano, & Tantillo, 2004). These methods include the development of conserved mitochondrial or nuclear DNA primers for PCR amplication, or alternatively, species-specic primers which have been shown to be suitable for the detection of meat adulteration due to a specic target sequence that can be detected in sequences of different

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origin without the need of further sequencing or digestion of the PCR products (Herman, 2001; Rodriguez et al., 2004). Although numerous studies have been devoted to identication of mammalian species, studies for detecting biological materials derived from avian species are scarce. The latter have mainly applied DNA-based techniques, such as specic PCR to authenticate ducks, geese, chickens and ostriches (Donne, Laudit, & Hanni, 2001; Hopwood, Fairbrother, Lockley, & Bardsley, 1999) or quail, pheasant, partridge and guinea fowl (Rojas et al., 2009), DNA hybridization for identifying chicken (Chikuni, Ozutsumi, Koishikawa, & Kato 1990), PCRRFLP (Girish et al., 2007; Meyer, Hoefelein, Luethy, & Candrian, 1995) and specic PCR (Behrens, Unthan, Bronkmann, Buchholz, & Latus, 1999) for identication of chicken, duck, turkey, guinea fowl and quail as well as RAPDPCR for differentiation of chicken, duck, turkey and goose (Calvo, Zaragoza, & Osta, 2001). Furthermore, in the majority of these studies, mitochondrial Cyt b gene was used as the molecular marker. The special place in modern eating habits that poultry meat holds, the high commercial value of some poultry products such as the meat of partridge or pheasant and the adulteration incidents of high with low value meat, forced us to attempt the development of a reliable method of tracing the origin of avian meat and avian meat products. Specically, with this study we aimed to develop a rapid, accurate, low-cost and easy-to-apply and interpret method of identication of both raw and processed meat from reared and game avian species, by applying a PCRRFLP technique on mitochondrial genes. 2. Materials and methods 2.1. Meat samples Muscle tissue samples from ten poultry and game bird species were collected and stored at 20 C till further treatment. The tested species were: chicken (Gallus gallus domesticus), turkey (Meleagris gallopavo), ostrich (Struthio camelus), duck (Anas platyrhynchos), goose (Anser anser), quail (Coturnix coturnix), rock partridge (Alectoris graeca), pheasant (Phasianus colchicus), eurasian woodcock (Scolopax rusticola), song thrush (Turdus philomelos). All were purchased from the local market in Larissa, Greece and initially identied according to standard species characteristics. Ten individuals at least were analyzed for each species in order to check for possible intraspecies polymorphism of the molecular markers. In addition, we analyzed food products of some of the above poultry species, subjected to various cooking methods or technological processes inherent to the meat sector such as roasted chicken and quail, raw and roasted chicken burgers, croquettes and Greek traditional roasted and chopped chicken meat (chicken gyros), roasted roll of turkey and fried turkey schnitzels, industrially processed chicken or turkey meat (salami products), boiled duck and pheasant, raw and fried ostrich sausages. Each sample was prepared and analyzed in triplicate. Furthermore, in order to identify possible contaminants of mammalian meat in poultry products, raw meat of beef, pig and sheep was analyzed. 2.2. DNA extraction The raw meat was chopped with sterile surgical blade. Representative portions of 0.2 g of each sample were homogenized using 0.5 ml TNES-Urea buffer (10 mM Tris pH 7.5, 125 mM NaCl, 10 mM EDTA, 0.5% SDS, 4 M Urea) containing 500 lg/ml proteinase K. The sample was incubated for 1 h at 55 C and subsequently was extracted twice with phenol: chloroform: isoamyl alcohol (25:24:1) and once with chloroform: isoamyl alcohol (24:1). Precipitation of DNA was performed by adding 50 ll of NaAc (3 M) and 1.2 ml

of ice-cold absolute ethanol and incubating at 20oC for 20 min. Genomic DNA was either collected by spooling with a pipette or precipitated by centrifugation for 10 min at full speed. In any case, genomic DNA was washed with 70% ethanol, dried at 37oC and resuspended in 100 ll sterilized distilled water. The DNA concentration was estimated by absorbance at 260 nm. 2.3. PCR amplication Two different mtDNA segments were PCR amplied: 12S rRNA and Cyt b. For both segments the amplication was performed using universal primers as described by Kocher et al. (1989). For 12S rRNA the primer pair was L-1091: 50 -AAA AAG CTT CAA ACT GGG ATT AGA TAC CCC ACT AT-30 (forward primer) and H-1478: 50 -TGA CTG CAG AGG GTG ACG GGG CGG TGT GT-30 (reverse primer) and for Cyt b the primer pair was L-14841: 50 -AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA-30 (forward primer) and H-15149: 50 -CTG CAG CCC CTC AGA ATG ATA TTT GTC CTC A-3 (reverse primer). For both segments, PCR amplications were performed in a nal volume of 50 ll containing two units of Taq polymerase, 5 ll of 10 reaction buffer (500 mM KCl, 100 mM Tris, pH 9.0), 0.8 mM dNTPs, 50 pmol of each primer, 2.5 mM MgCl2 and 500 ng of DNA. Amplications were performed in a Master Cycler Gradient Thermocycler (Eppendorf) applying the same conditions for both segments, as follows: 5 min at 95 C for initial denaturation, followed by 35 cycles of amplication (40 s at 95 C, 40 s at 58 C and 40 s at 72 C) and nal extension at 72 C for 10 min. PCR products were analyzed by electrophoresis in 2% agarose gel with ethidium bromide staining. 2.4. Restriction fragment length polymorphism (RFLP) analysis For each species, available mitochondrial genome sequences for 12S rRNA and Cyt b segments were retrieved from NCBIs Organelles Genome Resources database (http://www.ncbi.nlm.nih.gov/ genomes/OrganelleResource.cgi?taxid=33208) and were aligned by Bioedit (Hall, 1999). After performing simulated restriction enzyme digestions with the REBsites program (http://tools.neb.com/ REBsites/index.php), 10 restriction enzymes (AciI, AluI, AvaII, DdeI, HaeIII, HinfI, HhaI, MboI, MseI, TaqI) were selected, based on the criterion that they can discriminate at least two species and tested on both PCR amplied mtDNA segments in the conditions recommended by the manufacturer. After digestions with each one of the enzymes, restriction fragments were separated on 8% polyacrylamide gel and visualized by the silver staining technique. 3. Results and discussion In the current study, an attempt was made to molecularly identify raw and processed meat available in Greek market, from 10 reared and game avian species. The approach taken was based on interspecies nucleotide polymorphism of some of the sites of the mitochondrial genome. DNA extraction results showed that both raw and processed poultry meat samples yielded enough mtDNA for PCR amplication. The universal primers used under the selected conditions, amplied successfully all ten avian and the three mammalian species, as well as the processed and cooked products. Agarose gel electrophoresis of the PCR amplied products resolved approximately 380 bp and 480 bp for the Cyt b and 12S rRNA segments, respectively (Figs. 1 and 2, samples P) with only slight size differences between species. Thermal denaturation of DNA depends on heating temperature and time (Partis et al., 2000). Nevertheless, DNA is a quite hot-tolerant molecule and thus it has a clear advantage compared with

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Fig. 1. Different electrophoretic patterns produced after the digestion of Cyt b PCR product by AciI for ten different avian species. V100: ladder 100 bp, 1. Chicken, 2. Turkey, 3. Duck, 4. Goose, 5. Pheasant, 6. Ostrich, 7. Quail, 8. Rock partridge, 9. Eurasian woodcock, 10. Song thrush, P: undigested chicken PCR product.

Fig. 2. Different electrophoretic patterns produced after the digestion of the 12S rRNA by AciI for eleven different meat species. M25: DNA ladder 25 bp, 1. Chicken, 2. Turkey, 3. Duck, 4. Goose, 5. Pheasant, 6. Rock partridge, 7. Eurasian woodcock, 8. Song thrush, 9. Quail, 10. Ostrich, 11. Pig, P: undigested chicken PCR product, M100: DNA ladder 100 bp.

proteins in the molecular identication of processed food. During this study we analysed food products processed either by industrial practices or by simple domestic or restaurant cooking (boiling/frying/baking). Although conventional cooking methods affected the quality of extracted DNA (it appeared more fragmented, with a smear, after agarose electrophoresis and ethidium bromide staining), they did not affect the PCR amplication procedure. Therefore, restriction proles from the cooked poultry meat samples were identical to those from the corresponding samples of fresh meat. The same was true for all processed products examined, except from two heavily processed (heat and mill) products from chicken and turkey for which only Cyt b segment gave a PCR product but not 12S rRNA. This may be due to the smaller PCR product amplied for Cyt b. Arslan, Ilhak, & Calicioglu (2006) argued that molecular identication of processed meats could be achieved with PCR amplication targeting small DNA fragments (ca. 200 bp). Each restriction enzyme used in this study produced diagnostic restriction proles, able to discriminate between at least two avian species with both mtDNA segments. For Cyt b, the maximum species differentiation was achieved with the application of AciI restriction enzyme, which produced different proles between turkey, goose, rock partridge and song thrush (Fig. 1). However, the 12S rRNA segment, digested by AciI, gave the best resolution, showing different diagnostic prole for each species, distinguishing accurately and reproducibly all 10 species at once (Fig. 2). Observed digestion fragment sizes (Table 1) were comparable to those calculated by the in silico digestion of PCR products. The same approach was applied to raw, processed and cooked samples, producing identical results (data not shown). In addition, no intraspecic polymorphism was detected, at least for the 10 samples analyzed within each species (data not shown). Further verication for the absence of AciI restriction sites intraspecies polymorphisms came from the in silico analysis of all available 12S rRNA sequences retrieved from Genbank (http://www.ncbi.nlm.nih.gov/Genbank/ index.html). The usage of universal primers and the unique restriction enzyme during the PCRRFLP analysis greatly facilitates the identication of avian species in products in which the species origin is not obvious as well as in meat mixtures. Universal primers reduce the time and cost of the procedure in comparison to approaches where species-specic primers are applied and multiple PCR reactions are performed for the species recognition. Additionally, digestion of the PCR product with only one enzyme is denitely much simpler and economical relatively to multiple digests or sequencing, without interfering with the resolution of the analysis. For example, in another study using the same molecular marker for the identication and differentiation of ve poultry species (chicken, duck, turkey, quail, guinea fowl), four different restriction enzymes (HinfI, Mph1103I, MvaI, and Eco47I) were applied during the PCRRFLP procedure (Girish et al., 2007).

Table 1 Restriction proles and observed restriction fragment sizes for 11 different species, resulting after the digestion of 12S rRNA by AciI. Species Chicken Turkey Duck Goose Phaesant Partridge Woodcock Song thrush Quail Ostrich Pig Restriction fragments (bp) 150 260 130 110 250 240 175 115 210 125 250 95 95 75 95 95 95 95 100 95 100 135 90 75 65 75 75 75 85 85 80 90 95 80 45 60 70 45 45 70 70 50 70 20 45 20 45 60 20 20 45 60 45 45 20 45 45 30 20 20 Number of fragments 6 5 8 7 5 5 6 7 6 7 4 Sum of fragments (bp) 480 495 470 475 485 475 490 495 500 490 500

20 45 20 40

20 20

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The applicability of this technique in the frame of identication admixtures of different meats was shown during a routine survey of avian meat products from the local market. The ability to molecularly distinguish different species is of great socio-economical and commercial importance and prevents food mislabelling and misdescription, particularly if the food has been processed removing the ability to distinguish one ingredient from another. Particularly, an

admixture of two different meats was detected in sausages labeled as ostrich and further investigation with the PCRRFLP approach described, revealed that ostrich meat was adulterated with pork meat (Fig. 3). Interestingly, the same admixture was found also in fried unlabeled, presumably ostrich sausages from a restaurant, suggesting that adulteration and/or mislabeling could be more generalized (data not shown). The latter was further straightening by another case where industrially processed meat, labeled as turkey, was proven to be pure chicken after PCR amplication of Cyt b and digestion by the restriction enzymes HaeIII and HinfI (Fig. 4). We concluded that 10 avian species are identiable on a practical basis using the PCRRFLP technique with the digestion of a segment of mitochondrial 12S rRNA gene by the AciI restriction enzyme. The results of the current study suggest that our method provides a simple, rapid and cheap alternative to conventional mtDNA analysis for routinely studying numerous samples, as in inspection programs, for species identication. Acknowledgement The project was nanced by the Postgraduate Course Biotechnology-Quality Assessment in Nutrition and the Environment of the Department of Biochemistry and Biotechnology, University of Thessaly, Greece. References
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Fig. 3. Detection of meat admixture. The RFLP patterns of the ostrich sausage and pig samples after the digestion of the 12S rRNA by AciI. 1. Ostrich sausage, 2. Raw pig meat, 3. Raw ostrich meat, M100: DNA ladder 100 bp.

Fig. 4. Restriction proles of Cyt b after digestions by HaeIII and HinfI in different samples: 1. Fresh chicken, 2. Fried chicken, 3. Chicken hamburger, 4. Industrially processed chicken, 5. Fresh turkey, 6. Turkey schnitzel, 7. Industrially processed turkey A, 8. Industrially processed turkey B.

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