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Identification of hare meat by a species-specific marker of mitochondrial origin
Meat Science 90 (2012) 836–841
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Identification of hare meat by a species-specific marker of mitochondrial origin Cristina G. Santos a, b, 1, Vitor S. Melo a, b, 1, Joana S. Amaral a, b, Letícia Estevinho b, M. Beatriz P.P. Oliveira a, Isabel Mafra a,⁎ a b
REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal Instituto Politécnico de Bragança, Campus de Sta. Apolónia, 5301-857 Bragança, Portugal
a r t i c l e
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Article history: Received 13 September 2011 Received in revised form 26 October 2011 Accepted 26 October 2011 Keywords: Hare meat Lepus Species identification Authenticity Real-time PCR EvaGreen
a b s t r a c t Meat species identification in food has gained increasing interest in recent years due to public health, economic and legal concerns. Following the consumer trend towards high quality products, game meat has earned much attention. The aim of the present work was to develop a DNA-based technique able to identify hare meat. Mitochondrial cytochrome b gene was used to design species-specific primers for hare detection. The new primers proved to be highly specific to Lepus species, allowing the detection of 0.01% of hare meat in pork meat by polymerase chain reaction (PCR). A real-time PCR assay with the new intercalating EvaGreen dye was further proposed as a specific and fast tool for hare identification with increased sensitivity (1 pg) compared to end-point PCR (10 pg). It can be concluded that the proposed new primers can be used by both species-specific end-point PCR or real-time PCR to accurately authenticate hare meat. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Food authenticity assessment is a very important issue in that it avoids unfair competition among producers and allows consumers to have accurate information about the products they purchase. Nowadays, in the meat industry, the substitution of higher commercial valued meats by low-priced ones and the fraudulent labelling of meat species is becoming a concern (Fajardo et al., 2010). In particular, products from game animals are considered to be susceptible targets for frauds as they generally command higher prices compared to other meats. Additionally, game meat consumption has increased in recent years due to several motivations, such as its particular taste and flavour, its healthier composition (with lower fat and cholesterol contents), the absence of antibiotics and anabolic steroids, and the attraction for some people of eating new and exotic delicacies (Fajardo et al., 2010). In Central Europe, brown hare (Lepus europaeus) is one of the most abundant small game species (Fettinger, Smulders, Lazar, Omurtag, & Paulsen, 2010). In the Iberian Peninsula, the Iberian hare (Lepus granatensis) and the brown hare are two species that have been identified (Alves, Ferrand, Suchentrunk, & Harris, 2003). Cape hare (Lepus capensis) among all Lepus species has the largest
⁎ Corresponding author. Tel.: + 351 222078902. E-mail address: [email protected] (I. Mafra). 1 These authors contributed equally to the work. 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.10.018
distribution, ranging from South Africa across large parts of the continent to East, West, and North Africa, further to the Middle East, Central Asia, and the Pacific seaboard. Several other Lepus species have been identified, but phylogenetic relationships and systematics of hares are not well understood (Ben Slimen et al., 2008). Molecular studies based on mitochondrial DNA reveal quite complex evolutionary scenarios, such as the introgressive hybridisation between brown hares and mountain hares (L. timidus) (Thulin, Fang, & Averianov, 2006) and between Iberian hares and mountain hares (Alves et al., 2003). Recent reports support the hypothesis of conspecificity between cape and brown hares suggesting that differentiation patterns were not higher than within the taxa, being attributed to geographic distances rather than the occurrence of two species-specific gene pools (Ben Slimen et al., 2006, 2008). The term “game” for culinary purposes is used to describe all birds and animals that are hunted for food (Cobos, Hoz, Cambero, & Ordofiez, 1995). Hare, as a game meat, has been used in many recipes, such as the typical Portuguese dishes “arroz de lebre” and “cabidela de lebre” having rice and hare as major ingredients. More recently, hare meat is used in some delicacies such as pâtés. Traditionally, hunted hares are bought whole, and are mainly sold entire, with hair, so the consumer can confirm its genuineness. Following the European Union food hygiene legislation, countries are allowed to issue national regulations for this particular sector of local supply of small quantities of food (European Commission, 2004). Consequently, in some European countries, hunters, under some provisions, able to put on the market not only eviscerated game carcasses, but also
C.G. Santos et al. / Meat Science 90 (2012) 836–841
portioned and packed meat (Fettinger et al., 2010). Considering the above reasons and the accessibility of hares, restricted to hunting season and animal, hare meat is prone to be fraudulently substituted. Therefore, to be able to authenticate hare meat, analytical methodologies are required to specifically and unequivocally identify it. Recently, DNA-based methods have been considered as essential tools for species identification in animal foods and feedstuffs. In particular, the polymerase chain reaction (PCR) technique using speciesspecific primers is extensively used because of its potential for simple, fast, specific and sensitive analysis, enabling the identification of species even in complex and processed foods (Bottero & Dalmasso, 2010; Mafra, Ferreira, & Oliveira, 2008). Most reported PCR applications for meat species identification have focused on domestic species like cattle, sheep, goat, domestic pig, turkey or chicken (Ballin, Vogensen, & Karlsson, 2009; Colgan et al., 2001; Girish et al., 2005; Mafra, Ferreira, Faria, & Oliveira, 2004; Mafra, Roxo, Ferreira, & Oliveira, 2007; Matsunaga et al., 1999; Soares, Amaral, Mafra, & Oliveira, 2010). In contrast, PCR assays dealing with game meat identification are less, but with an increasing trend in the past few years (Fajardo et al., 2010). New developments of quantitative analysis based on real-time PCR offer the potential for rapid results, high throughput, high sensitivity and opportunities for automation (López-Calleja et al., 2007a, b). In real-time PCR, the amplification of products is directly monitored along each amplification cycle, allowing the measurement of the PCR process at an early stage of the exponential phase and, therefore, providing a quantitative result. Data collection is achieved using fluorescent molecules able to provide a strong correlation and to measure minute amounts of different animal species (Fajardo et al., 2008; Mafra, Ferreira, & Oliveira, 2008). Real-time PCR is based on the use of fluorescent compounds, which can be either a general dye, such as SYBR Green, that intercalates in DNA molecules, or different probebased systems, which confer an added specificity to the reaction. In both cases, fluorescence is proportional to the amount of DNA present (Dooley, Paine, Garrett, & Brown, 2004; Fajardo et al., 2008; Mafra, Ferreira, & Oliveira, 2008). The use of DNA binding dyes offers a suitable and less expensive alternative to real-time PCR since it is a more flexible method without requiring an individual probe design and optimisation steps (Fajardo et al., 2008). The use of SYBR Green real-time PCR has been successfully applied to the identification and quantification of game meats, namely red deer, fallow deer and roe deer, in meat mixtures (Fajardo et al., 2008). More recently, EvaGreen has been reported as a novel DNA intercalating dye, more stable and sensitive than SYBR Green (Wang, Chen, & Xu, 2006). The enhanced fluorescent EvaGreen dye can be used at higher concentrations than SYBR Green I, generating greater fluorescent signals, increased sensitivity and excellent stability without causing PCR inhibition. The application of real-time PCR with EvaGreen fluorescent dye has been reported for the identification of cervidae species (Chen et al., 2009). The aim of the present work was to develop a highly specific and sensitive PCR technique to identify and authenticate hare meat. For this purpose, hare specific primers were designed targeting the cytochrome b (cytb) mitochondrial gene to amplify a 127 bp fragment. The new primers were used to develop a simple species-specific PCR approach to identify hare meat. By the use of real-time PCR with the new generation dye EvaGreen, a highly sensitive technique for hare detection was proposed. 2. Materials and methods 2.1. Samples Samples of authentic hare meat were acquired from local hunters and retail market. Each sample consisted on an entire animal, not eviscerated, with hair, to confirm its authenticity. For each animal, fresh muscle portions were selected and minced in a blender.
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Reference binary mixtures containing known proportions of hare meat in pork meat (0.01%, 0.1%, 0.5%, 1%, 2.5%, 5%, 10% and 20% (w/w)) were prepared by homogenising both minced meats with a blender to a final weight of 100 g. To avoid contamination, each mixture was processed separately using different material and different blender containers previously treated with DNA decontaminator solution. A wide range of non-target animal species was also included in the study for specificity assays, namely rabbit (Oryctolagus cuniculus), partridge (Alectoris spp.), red legged partridge (Alectoris rufa), pheasant (Phasianus colchinus), quail (Coturnix coturnix), turkey (Meleagris gallopavo), chicken (Gallus gallus), duck (Anas platyrhynchos), ostrich (Struthio camelus), beef (Bos taurus), sheep (Ovis aries), goat (Capra hircus), pig (Sus scrofa domestica), wild boar (Sus scrofa scrofa) and red deer (Cervus elaphus). All samples were extracted immediately or stored at −20 °C to prevent enzymatic DNA degradation. 2.2. DNA extraction DNA was extracted using the Wizard method as described by Mafra, Silva, Moreira, Ferreira da Silva, and Oliveira (2008) with minor modifications. Briefly, 100 mg of ground and homogenised samples were transferred to a 2 mL sterile reaction tube followed by the addition of 860 μL of TNE extraction buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% SDS), 100 μL of 5 M guanidine hydrochloride solution and 40 μL proteinase K solution (20 mg/mL). After the incubation at 60 °C for 3 h, with occasional stirring, the suspension was centrifuged (15 min, 18,514 g) and 500 μL of the supernatant was mixed with 1 mL of Wizard DNA purification resin (Promega, Madison, WI, USA). The mixture was eluted through a column and the resin washed with 2 mL of isopropanol solution (80%, v/v). After drying the column, the DNA was eluted by centrifugation (1 min 10,000 g) with 100 μL of Tris–EDTA buffer (10 mM Tris, 1 mM EDTA) at 70 °C to a new reaction tube. The extractions were performed in duplicate assays for each binary mixture. The quality of extracted DNA was analysed by electrophoresis in a 1.0% agarose gel containing Gel Red 1x (Biotium, Hayward, CA, USA) for staining and carried out in TAE buffer (40 mM Tris–acetate, 1 mM EDTA) for 40 min at 120 V. The agarose gel was visualised under UV light and a digital image was obtained using a Kodak Digital ScienceTM DC120 (Rochester, NY, USA). 2.3. DNA quantification and purity The DNA was quantified by spectrophotometry using a Shimadzu UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) as described by Somma (2006). The DNA concentration was determined by UV absorbance at 260 nm (1 absorbance unit corresponds to 50 μg/mL of dsDNA). The purity of the extracted DNA was determined by the ratio of the absorbances at 260 and 280 nm. 2.4. Target gene selection and oligonucleotide primers Oligonucleotide primers to identify hare meat were designed on the basis of the mitochondrial cytb gene sequence from various animal and plant species available in the NCBI (National Center for Biotechnology Information) Genbank database. The specific primers were designed using the software Primer3 Output designing tool (http://frodo.wi.mit.edu/primer3/). The nucleotide sequence was submitted to the basic local alignment search tool BLAST (http:// blast.ncbi.nlm.nih.gov/Blast.cgi), which identifies regions of local similarity among homologue sequences of different species and calculates the statistical significance of the matches (Altschul, Gish, Miller, Myers, & Lipman, 1990).
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facility (STABVIDA, Lisbon, Portugal) for sequencing. The method of choice for small PCR fragments was the direct sequencing of both strands in opposite directions, allowing the production of two complementary sequences with good quality.
Table 1 Oligonucleotide primers. Primer
Target gene
Lep-F
Cytb
Sequence (5' → 3')
Amplicon Reference (bp)
ATA CAT GTA GGC CGT GGA 127 ATC TAC Lep-R TTT GTC CTC ATG GGA GGA CGT A 18SEU-F 18S rRNA TCT GCC CTA TCA ACT TTC GAT 140 GG 18SEU-R TAA TTT GCG CGC CTG CTG
This work*
2.7. EvaGreen real-time PCR Fajardo et al. (2008)
* Genbank accession number AJ534882.
As positive amplification control of PCR experiments, previously reported universal primers targeting a conserved 18S rRNA gene fragment in all eukaryotic cells, were used (Fajardo et al., 2008). All the primers were synthesised by Eurofins MWG Operon (Ebersberg, Germany) (Table 1). 2.5. End-point PCR Amplifications by end-point PCR were performed in a total reaction volume of 25 μL containing 2 μL of DNA extract (20 ng), 67 mM Tris– HCl (pH 8.8), 16 mM (NH4)2SO4, 0.01% Tween 20, 200 μM dNTP (Bioron,, Ludwigshafen, Germany), MgCl , 200 nM or 500 nM of each primer for hare (Lep-F/Lep-R) or eukaryotic sequences (18SEU-F/ 18SEU-R), respectively (Table 1), 2.5 mM or 1.5 mM of MgCl2 for hare or eukaryotic DNA, respectively, and 1 U of SuperHot Taq DNA polymerase (Genaxxon Bioscience GmbH, Ulm, Germany). The reactions were performed in a thermal cycler MJ Mini (Bio-Rad Laboratories, Hercules, CA, USA) using the following program: initial denaturation at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 65 °C or 60 °C for hare or eukaryotic DNA, respectively, for 30 s and 72 °C for 45 s; and a final extension at 72 °C for 5 min. The amplified fragments were analysed by electrophoresis in a 2.0% agarose gel containing Gel Red 1x (Biotium, Hayward, CA, USA) for staining and carried out in TAE buffer (40 mM Tris–acetate, 1 mM EDTA) for 60 min at 120 V. The agarose gel was visualised under UV light and a digital image was obtained using a Kodak Digital Science™ DC120 (Rochester, NY, USA). Each extract was amplified at least in duplicate assays. 2.6. Sequencing PCR products To confirm the identity of the PCR products obtained with the Lep-F/Lep-R primers, the expected 127 bp fragments were sequenced. PCR products were purified in Cut&Spin DNA gel extraction columns (GRISP Research Solutions, Porto, Portugal) to remove interfering components. The purified products were sent to a specialised research
M
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The amplifications by real-time PCR were carried out in 20 μL of total reaction volume containing 2 μL of DNA extract (100 ng to 0.1 pg), 1× of SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and 300 nM of each primer Lep-F/Lep-R (Table 1). The real-time PCR assays were performed on a fluorometric thermal cycler CFX96 Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using the following conditions: 95 °C for 5 min; 45 cycles at 95 °C for 10 s and 65 °C for 30 s, with collection of fluorescence signal at the end of each cycle. Data was collected and processed using the software Bio-Rad CFX Manager 2.0 (Bio-Rad Laboratories, Hercules, CA, USA). For melting curve analysis, PCR products were denatured at 95 °C for 1 min and then annealed at 65 °C for 5 min in order to allow the correct annealing of the DNA duplexes. These two steps were followed by melting curves ranging from 65.0 to 95.0 °C with temperature increments of 0.2 °C every 10 s. The fluorescence data were acquired at the end of each melting temperature. The data collected were processed using the Precision Melt Analysis Software 1.0 (Bio-Rad Laboratories, Hercules, CA, USA). Each reference mixture was analysed in replicates (n = 4) in two independent assays. 3. Results and discussion 3.1. Species-specific PCR This paper reports the specific identification of brown hare based on the design of new primers (Lep-F/Lep-R) targeting the mitochondrial cytb gene of Lepus europaeus. The BLAST results for the expected amplified fragment of 127 bp confirmed 100% specificity for Lepus europaeus, while other Lepus species (L. capensis, L. townsendii, L. yarkandensis, L. oiostolus, L. granatensis, L. brachyurus and L. hainurus) showed a maximum identity from 94 to 96%. The cytb gene was chosen for the present work since it is the most studied gene for phylogeny, being the most targeted gene for molecular identification studies (Teletchea, Maudet, & Hanni, 2005). To verify the specificity of the proposed primers to Lepus species and against other animal species commonly used as food, including game meat, the DNA of wild boar, duck, partridge, quail, rabbit, pheasant, red deer, wild rabbit, cow, chicken, turkey, ostrich, goat and sheep meats was used for PCR amplification. In Fig. 1, the results clearly indicate that Lep-F/Lep-R primers were able to amplify the
9 10 11 12 13 14 15 16
C
127 bp
Fig. 1. Agarose gel electrophoresis of PCR products targeting Lepus cytb gene. M - 100 bp ladder (Bioron, Ludwigshafen, Germany); lane 1 - wild boar; lane 2 - duck; lane 3 - partridge; lane 4 - hare; lane 5 - quail; lane 6 - rabbit; lane 7 - pheasant; lane 8 – red deer; lane 9 - wild rabbit; lane 10 - cow; lane 11 - chicken; lane 12 - turkey; lane 13 - sheep; lane 14: - goat; lane 15 - ostrich; lane 16 - pork; lane C - negative control.
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Fig. 2. Sequence alignment of mitochondrial cytb entry (AJ534882) with the obtained fragment using primers Lep-F/Lep-R. In italics is represented the sequenced fragment. Underlined is the region of primer annealing. The grey letters represent the mismatching bases.
expected 127 bp of cytb from L. europaeus species. All the extracts were positively amplified to the nuclear 18S rRNA gene for eukaryotic DNA with the universal primers (18SEU-F/18SEU-R), confirming that they contained amplifiable DNA (data not shown). To confirm the homology of the obtained Lepus fragment, the sequenced DNA was aligned with the expected cytb sequence (Fig. 2). The results revealed the occurrence of 7 mismatches, which gives a maximum identity of 95% against the cytb gene of L. europaeus. Performing a new BLAST for the obtained sequence, the results showed a 100% of homology for L. granatensis, which is a species also found in several regions of Portugal and Spain (Alves et al., 2003). Using the Primer-BLAST tool for the proposed primers, the results confirm the complete matching for several cytb entries of L. granatensis, L. europaeus and L. capensis. Many other Lepus species are also probably targeted since they include only one mismatch in one of the primers. This finding supports a closed phylogenetic relationship among several Lepus species (Alves et al., 2003; Ben Slimen et al., 2006; 2008; Pérez-Suarez, Palacios, & Boursot, 1994). The optimised PCR conditions using the binary model mixtures containing known amounts of hare meat in pork meat gave the relative limit of detection of 0.01% of hare meat (Fig. 3). The sensitivity of hare specific PCR assay was determined by amplification of a DNA extract of hare 10-fold serially diluted. The results can be observed in Fig. 4, where the amplification of the lowest level of 10 pg for hare DNA can be seen.
3.2. Real-time PCR Following the successful application of Lep-F/Lep-R primers to species-specific PCR detection, a real-time PCR assay was proposed to identify hare meat. Real-time PCR data collection was achieved
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with the new fluorescent intercalating dye EvaGreen, as a more sensitive and alternative approach than the SYBR Green dye. The results of real-time PCR amplification of hare DNA from 10-fold serially diluted extracts are presented in Fig. 5. The amplification curves (Fig. 5a) indicate the adequacy of the proposed primers for real-time PCR that was confirmed with the respective standard curve (Fig. 5b). The PCR efficiency was very close to the ideal value of 100% that corresponds to a slope of − 3.32, and all data were highly correlated (R 2 > 0.99). The obtained parameters show the appropriateness of the assay for quantitative measurements. The limit of detection was 1 pg, which was improved compared to end-point PCR (10 pg) (Fig. 4). To confirm the specificity of amplified products, a melting curve analysis was performed right after real-time amplification curves. In Fig. 5c, it can be verified that all products have the same melting temperature of 78.2 °C, without showing any evidence of primer dimer or other different fragments. The use of EvaGreen dye contributed to the robust PCR signals obtained as well as strong sharp DNA melt peaks. The relatively high dye concentration in quantitative PCR eliminates dye redistribution problems, making it suitable for fast cycling, high through put and high resolution melting application (Mao, Leung, & Xin, 2007).
4. Conclusions In the present work, two new PCR approaches targeting the mitochondrial cytb gene to identify hare species were proposed. The species-specific PCR assay based on end-point PCR proved to be highly specific and sensitive to hare, reaching a relative level of detection of 0.01% of hare meat and an absolute limit of detection of 10 pg of hare DNA. The second approach based on real-time PCR with the new intercalating EvaGreen dye was also a highly specific technique
C M
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127 bp
Fig. 3. Agarose gel electrophoresis of PCR products targeting Lepus cytb gene of binary reference mixtures of hare in pork meat. M, 100 bp ladder (Bioron, Ludwigshafen, Germany); lane 1, 100% pork meat; lanes 2–9, different proportions of hare in pork meat; C, negative control.
1 pg
0.5 0.1 0.01%
10 pg
1
1 ng
2.5
10 ng
5
0.1 ng
127 bp 20 10
Fig. 4. Agarose gel electrophoresis of PCR products targeting Lepus cytb gene of different amounts of DNA (lanes 1–7). M, 100 bp ladder (Bioron, Ludwigshafen, Germany); C, negative control.
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a 100 ng 10 ng 1 ng 0.1 ng 10 pg 1 pg 0.1 pg
b
c
Fig. 5. Real-time PCR amplification with EvaGreen intercalating dye targeting the cytb gene of hare. (a) Amplification curves; (b) calibration curve; and (c) melting curves.
for hare identification with a higher sensitivity than end-point PCR (1 pg). The real-time PCR assay proved adequate for quantitative analysis and high throughput hare identification in inspection programs, thus enforcing labelling regulations in the meat industry.
Acknowledgements The authors acknowledge the financial support of U. Porto/Santander Totta “Projectos pluridisciplinares 2010”. This work has also been supported by Fundação para a Ciência e a Tecnologia (FCT) through grant no. PEst-C/EQB/LA0006/2011. The authors are grateful to Prof.
Vasco Cadavez from the Animal Science Department of Polytechnic Institute of Bragança for the supply of samples. References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410. Alves, P. C., Ferrand, N., Suchentrunk, F., & Harris, D. J. (2003). Ancient introgression of Lepus timidus mtDNA into L. granatensis and L. europaeus in the Iberian Peninsula. Molecular Phylogenetics and Evolution, 27, 70–80. Ballin, N. Z., Vogensen, F. K., & Karlsson, A. H. (2009). Species determination – can we detect and quantify meat adulteration? Meat Science, 83, 165–174. Ben Slimen, H., Suchentrunk, Memmi, A., Sert, H., Kryger, U., Alves, P. C., et al. (2006). Evolutionary relationships among hares from North Africa (Lepus sp. or Lepus
C.G. Santos et al. / Meat Science 90 (2012) 836–841 spp.) cape hares (L. capensis) from South Africa, and brown hares (L. europaeus) as inferred from mtDNA PCR-RFLP and allozyme data. Journal of Zoological Systematics and Evolutionary Research, 44, 88–99. Ben Slimen, H., Suchentrunk, F., Stamatis, C., Mamuris, Z., Sert, H., Alves, P. C., et al. (2008). Population genetics of cape and brown hares (Lepus capensis and L. europaeus): A test of Petter's hypothesis of conspecificity. Biochemical Systematics and Ecology, 36, 22–39. Bottero, M. T., & Dalmasso, A. (2010). Animal species identification in food products: Evolution of biomolecular methods. The Veterinary Journal. doi:10.1016/j.tvjl.2010.09.024. Chen, Y., Wu, Y., Wang, J., Xu, B., Zhong, Z., & Xia, J. (2009). Identification of cervidae DNA in feedstuff using a real-time polymerase chain reaction method with the new fluorescence intercalating dye EvaGreen. Journal of AOAC International, 92, 175–180. Cobos, A., Hoz, L., Cambero, M. I., & Ordofiez, J. A. (1995). Chemical and fatty acid composition of meat from spanish wild rabbits and hares. Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 200, 182–185. Colgan, S., O'Brien, L., Maher, M., Shilton, N., McDonnell, K., & Ward, S. (2001). Development of a DNA-based assay for species identification in meat and bone meal. Food Research International, 34, 409–414. Dooley, J. J., Paine, K. E., Garrett, S. D., & Brown, H. M. (2004). Detection of meat species using TaqMan real-time PCR assays. Meat Science, 68, 431–438. European Commission (2004). Regulation (EC) No. 852/2004 of the European Parliament and the Council of 29th of April 2004 on the hygiene of foodstuffs. Official Journal of the European Union, Brussels O.J. L 138/1. Fajardo, V., González, I., Martín, I., Rojas, M., García, T., & Martín, R. (2010). A review of current PCR-based methodologies for the authentication of meats from game animal species. Trends in Food Science and Technology, 21, 408–421. Fajardo, V., González, I., Martín, I., Rojas, M., Hernández, P. E., García, T., et al. (2008). Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures. Meat Science, 79, 289–298. Fettinger, V., Smulders, F. J. M., Lazar, P., Omurtag, I., & Paulsen, P. (2010). Lesions in thighs from hunted Brown Hares (Lepus europaeus) and microflora under vacuum-packaging storage. European Journal of Wildlife Research, 56, 943–947. Girish, P. S., Anjaneyulu, A. S. R., Viswas, K. N., Shivakumar, B. M., Anand, M., Patel, M., et al. (2005). Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene. Meat Science, 70, 107–112. López-Calleja, I., González, I., Fajardo, V., Martín, I., Hernández, P. E., García, T., et al. (2007). Quantitative detection of goats’ milk in sheep's milk by real-time PCR. Food Control, 18, 1466–1473.
841
López-Calleja, I., González, I., Fajardo, V., Martín, I., Hernández, P. E., García, T., et al. (2007). Real-time TaqMan PCR for quantitative detection of cows’ milk in ewes’ milk mixtures. International Dairy Journal, 17, 729–736. Mafra, I., Ferreira, I. M. P. L. V. O., Faria, M. A., & Oliveira, B. P. P. (2004). A novel approach to the quantification of bovine milk in ovine cheeses using a duplex polymerase chain reaction method. Journal of Agricultural and Food Chemistry, 52, 4943–4947. Mafra, I., Ferreira, I. M. P. L. V. O., & Oliveira, M. B. P. P. (2008). Food authentication by PCR-based methods. European Food Research and Technology, 227, 649–665. Mafra, I., Roxo, A., Ferreira, I. M. P. L. V. O., & Oliveira, M. B. P. P. (2007). A duplex polymerase chain reaction for the quantitative detection of cows' milk in goats' milk cheese. International Dairy Journal, 17, 1132–1138. Mafra, I., Silva, S. A., Moreira, E. J. M. O., Ferreira da Silva, C. S., & Oliveira, M. B. P. P. (2008). Comparative study of DNA extraction methods for soybean derived food products. Food Control, 19, 1183–1190. Mao, F., Leung, W. -Y., & Xin, X. (2007). Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology, 7, 76. Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J., et al. (1999). A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science, 51, 143–148. Pérez-Suarez, G., Palacios, F., & Boursot, P. (1994). Speciation and paraphyly in western mediterranean hares (Lepus castroviejoi, L. europaeus, L. granatensis, and L. capensis) revealed by mithocondrial DNA phylogeny. Biochemical Genetics, 32, 423–436. Soares, S., Amaral, J. S., Mafra, I., & Oliveira, M. B. P. P. (2010). Quantitative detection of pork's meat by duplex PCR. Meat Science, 85, 531–536. Somma, M. (2006). Extraction and purification of DNA (Session 4). In M. Querci, M. Jermini, & G. V. Eede (Eds.), Training course on The Analysis of Food Samples for the Presence of Genetically Modified Organisms. European Commission DG-JRC http:// mbg.jrc.ec.europa.eu/capacitybuilding/manuals/Manual%20EN/User%20Manual%20 EN%20full.pdf Teletchea, F., Maudet, C., & Hanni, C. (2005). Food and forensic molecular identification: update and challenges. Trends in Biotechnology, 23, 359–366. Thulin, C. -G., Fang, M., & Averianov, A. O. (2006). Introgression from Lepus europeaus to L. timidus in Russia revealed by mitochondrial single nucleotide polymorphisms and nuclear microsatellites. Hereditas, 143, 68–76. Wang, W., Chen, K., & Xu, C. (2006). DNA quantification using EvaGreen and a real-time PCR instrument. Analytical Biochemistry, 356, 303–305.
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Meat Science journal homepage: www.elsevier.com/locate/meatsci
Identification of hare meat by a species-specific marker of mitochondrial origin Cristina G. Santos a, b, 1, Vitor S. Melo a, b, 1, Joana S. Amaral a, b, Letícia Estevinho b, M. Beatriz P.P. Oliveira a, Isabel Mafra a,⁎ a b
REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal Instituto Politécnico de Bragança, Campus de Sta. Apolónia, 5301-857 Bragança, Portugal
a r t i c l e
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Article history: Received 13 September 2011 Received in revised form 26 October 2011 Accepted 26 October 2011 Keywords: Hare meat Lepus Species identification Authenticity Real-time PCR EvaGreen
a b s t r a c t Meat species identification in food has gained increasing interest in recent years due to public health, economic and legal concerns. Following the consumer trend towards high quality products, game meat has earned much attention. The aim of the present work was to develop a DNA-based technique able to identify hare meat. Mitochondrial cytochrome b gene was used to design species-specific primers for hare detection. The new primers proved to be highly specific to Lepus species, allowing the detection of 0.01% of hare meat in pork meat by polymerase chain reaction (PCR). A real-time PCR assay with the new intercalating EvaGreen dye was further proposed as a specific and fast tool for hare identification with increased sensitivity (1 pg) compared to end-point PCR (10 pg). It can be concluded that the proposed new primers can be used by both species-specific end-point PCR or real-time PCR to accurately authenticate hare meat. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Food authenticity assessment is a very important issue in that it avoids unfair competition among producers and allows consumers to have accurate information about the products they purchase. Nowadays, in the meat industry, the substitution of higher commercial valued meats by low-priced ones and the fraudulent labelling of meat species is becoming a concern (Fajardo et al., 2010). In particular, products from game animals are considered to be susceptible targets for frauds as they generally command higher prices compared to other meats. Additionally, game meat consumption has increased in recent years due to several motivations, such as its particular taste and flavour, its healthier composition (with lower fat and cholesterol contents), the absence of antibiotics and anabolic steroids, and the attraction for some people of eating new and exotic delicacies (Fajardo et al., 2010). In Central Europe, brown hare (Lepus europaeus) is one of the most abundant small game species (Fettinger, Smulders, Lazar, Omurtag, & Paulsen, 2010). In the Iberian Peninsula, the Iberian hare (Lepus granatensis) and the brown hare are two species that have been identified (Alves, Ferrand, Suchentrunk, & Harris, 2003). Cape hare (Lepus capensis) among all Lepus species has the largest
⁎ Corresponding author. Tel.: + 351 222078902. E-mail address: [email protected] (I. Mafra). 1 These authors contributed equally to the work. 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.10.018
distribution, ranging from South Africa across large parts of the continent to East, West, and North Africa, further to the Middle East, Central Asia, and the Pacific seaboard. Several other Lepus species have been identified, but phylogenetic relationships and systematics of hares are not well understood (Ben Slimen et al., 2008). Molecular studies based on mitochondrial DNA reveal quite complex evolutionary scenarios, such as the introgressive hybridisation between brown hares and mountain hares (L. timidus) (Thulin, Fang, & Averianov, 2006) and between Iberian hares and mountain hares (Alves et al., 2003). Recent reports support the hypothesis of conspecificity between cape and brown hares suggesting that differentiation patterns were not higher than within the taxa, being attributed to geographic distances rather than the occurrence of two species-specific gene pools (Ben Slimen et al., 2006, 2008). The term “game” for culinary purposes is used to describe all birds and animals that are hunted for food (Cobos, Hoz, Cambero, & Ordofiez, 1995). Hare, as a game meat, has been used in many recipes, such as the typical Portuguese dishes “arroz de lebre” and “cabidela de lebre” having rice and hare as major ingredients. More recently, hare meat is used in some delicacies such as pâtés. Traditionally, hunted hares are bought whole, and are mainly sold entire, with hair, so the consumer can confirm its genuineness. Following the European Union food hygiene legislation, countries are allowed to issue national regulations for this particular sector of local supply of small quantities of food (European Commission, 2004). Consequently, in some European countries, hunters, under some provisions, able to put on the market not only eviscerated game carcasses, but also
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portioned and packed meat (Fettinger et al., 2010). Considering the above reasons and the accessibility of hares, restricted to hunting season and animal, hare meat is prone to be fraudulently substituted. Therefore, to be able to authenticate hare meat, analytical methodologies are required to specifically and unequivocally identify it. Recently, DNA-based methods have been considered as essential tools for species identification in animal foods and feedstuffs. In particular, the polymerase chain reaction (PCR) technique using speciesspecific primers is extensively used because of its potential for simple, fast, specific and sensitive analysis, enabling the identification of species even in complex and processed foods (Bottero & Dalmasso, 2010; Mafra, Ferreira, & Oliveira, 2008). Most reported PCR applications for meat species identification have focused on domestic species like cattle, sheep, goat, domestic pig, turkey or chicken (Ballin, Vogensen, & Karlsson, 2009; Colgan et al., 2001; Girish et al., 2005; Mafra, Ferreira, Faria, & Oliveira, 2004; Mafra, Roxo, Ferreira, & Oliveira, 2007; Matsunaga et al., 1999; Soares, Amaral, Mafra, & Oliveira, 2010). In contrast, PCR assays dealing with game meat identification are less, but with an increasing trend in the past few years (Fajardo et al., 2010). New developments of quantitative analysis based on real-time PCR offer the potential for rapid results, high throughput, high sensitivity and opportunities for automation (López-Calleja et al., 2007a, b). In real-time PCR, the amplification of products is directly monitored along each amplification cycle, allowing the measurement of the PCR process at an early stage of the exponential phase and, therefore, providing a quantitative result. Data collection is achieved using fluorescent molecules able to provide a strong correlation and to measure minute amounts of different animal species (Fajardo et al., 2008; Mafra, Ferreira, & Oliveira, 2008). Real-time PCR is based on the use of fluorescent compounds, which can be either a general dye, such as SYBR Green, that intercalates in DNA molecules, or different probebased systems, which confer an added specificity to the reaction. In both cases, fluorescence is proportional to the amount of DNA present (Dooley, Paine, Garrett, & Brown, 2004; Fajardo et al., 2008; Mafra, Ferreira, & Oliveira, 2008). The use of DNA binding dyes offers a suitable and less expensive alternative to real-time PCR since it is a more flexible method without requiring an individual probe design and optimisation steps (Fajardo et al., 2008). The use of SYBR Green real-time PCR has been successfully applied to the identification and quantification of game meats, namely red deer, fallow deer and roe deer, in meat mixtures (Fajardo et al., 2008). More recently, EvaGreen has been reported as a novel DNA intercalating dye, more stable and sensitive than SYBR Green (Wang, Chen, & Xu, 2006). The enhanced fluorescent EvaGreen dye can be used at higher concentrations than SYBR Green I, generating greater fluorescent signals, increased sensitivity and excellent stability without causing PCR inhibition. The application of real-time PCR with EvaGreen fluorescent dye has been reported for the identification of cervidae species (Chen et al., 2009). The aim of the present work was to develop a highly specific and sensitive PCR technique to identify and authenticate hare meat. For this purpose, hare specific primers were designed targeting the cytochrome b (cytb) mitochondrial gene to amplify a 127 bp fragment. The new primers were used to develop a simple species-specific PCR approach to identify hare meat. By the use of real-time PCR with the new generation dye EvaGreen, a highly sensitive technique for hare detection was proposed. 2. Materials and methods 2.1. Samples Samples of authentic hare meat were acquired from local hunters and retail market. Each sample consisted on an entire animal, not eviscerated, with hair, to confirm its authenticity. For each animal, fresh muscle portions were selected and minced in a blender.
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Reference binary mixtures containing known proportions of hare meat in pork meat (0.01%, 0.1%, 0.5%, 1%, 2.5%, 5%, 10% and 20% (w/w)) were prepared by homogenising both minced meats with a blender to a final weight of 100 g. To avoid contamination, each mixture was processed separately using different material and different blender containers previously treated with DNA decontaminator solution. A wide range of non-target animal species was also included in the study for specificity assays, namely rabbit (Oryctolagus cuniculus), partridge (Alectoris spp.), red legged partridge (Alectoris rufa), pheasant (Phasianus colchinus), quail (Coturnix coturnix), turkey (Meleagris gallopavo), chicken (Gallus gallus), duck (Anas platyrhynchos), ostrich (Struthio camelus), beef (Bos taurus), sheep (Ovis aries), goat (Capra hircus), pig (Sus scrofa domestica), wild boar (Sus scrofa scrofa) and red deer (Cervus elaphus). All samples were extracted immediately or stored at −20 °C to prevent enzymatic DNA degradation. 2.2. DNA extraction DNA was extracted using the Wizard method as described by Mafra, Silva, Moreira, Ferreira da Silva, and Oliveira (2008) with minor modifications. Briefly, 100 mg of ground and homogenised samples were transferred to a 2 mL sterile reaction tube followed by the addition of 860 μL of TNE extraction buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% SDS), 100 μL of 5 M guanidine hydrochloride solution and 40 μL proteinase K solution (20 mg/mL). After the incubation at 60 °C for 3 h, with occasional stirring, the suspension was centrifuged (15 min, 18,514 g) and 500 μL of the supernatant was mixed with 1 mL of Wizard DNA purification resin (Promega, Madison, WI, USA). The mixture was eluted through a column and the resin washed with 2 mL of isopropanol solution (80%, v/v). After drying the column, the DNA was eluted by centrifugation (1 min 10,000 g) with 100 μL of Tris–EDTA buffer (10 mM Tris, 1 mM EDTA) at 70 °C to a new reaction tube. The extractions were performed in duplicate assays for each binary mixture. The quality of extracted DNA was analysed by electrophoresis in a 1.0% agarose gel containing Gel Red 1x (Biotium, Hayward, CA, USA) for staining and carried out in TAE buffer (40 mM Tris–acetate, 1 mM EDTA) for 40 min at 120 V. The agarose gel was visualised under UV light and a digital image was obtained using a Kodak Digital ScienceTM DC120 (Rochester, NY, USA). 2.3. DNA quantification and purity The DNA was quantified by spectrophotometry using a Shimadzu UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) as described by Somma (2006). The DNA concentration was determined by UV absorbance at 260 nm (1 absorbance unit corresponds to 50 μg/mL of dsDNA). The purity of the extracted DNA was determined by the ratio of the absorbances at 260 and 280 nm. 2.4. Target gene selection and oligonucleotide primers Oligonucleotide primers to identify hare meat were designed on the basis of the mitochondrial cytb gene sequence from various animal and plant species available in the NCBI (National Center for Biotechnology Information) Genbank database. The specific primers were designed using the software Primer3 Output designing tool (http://frodo.wi.mit.edu/primer3/). The nucleotide sequence was submitted to the basic local alignment search tool BLAST (http:// blast.ncbi.nlm.nih.gov/Blast.cgi), which identifies regions of local similarity among homologue sequences of different species and calculates the statistical significance of the matches (Altschul, Gish, Miller, Myers, & Lipman, 1990).
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facility (STABVIDA, Lisbon, Portugal) for sequencing. The method of choice for small PCR fragments was the direct sequencing of both strands in opposite directions, allowing the production of two complementary sequences with good quality.
Table 1 Oligonucleotide primers. Primer
Target gene
Lep-F
Cytb
Sequence (5' → 3')
Amplicon Reference (bp)
ATA CAT GTA GGC CGT GGA 127 ATC TAC Lep-R TTT GTC CTC ATG GGA GGA CGT A 18SEU-F 18S rRNA TCT GCC CTA TCA ACT TTC GAT 140 GG 18SEU-R TAA TTT GCG CGC CTG CTG
This work*
2.7. EvaGreen real-time PCR Fajardo et al. (2008)
* Genbank accession number AJ534882.
As positive amplification control of PCR experiments, previously reported universal primers targeting a conserved 18S rRNA gene fragment in all eukaryotic cells, were used (Fajardo et al., 2008). All the primers were synthesised by Eurofins MWG Operon (Ebersberg, Germany) (Table 1). 2.5. End-point PCR Amplifications by end-point PCR were performed in a total reaction volume of 25 μL containing 2 μL of DNA extract (20 ng), 67 mM Tris– HCl (pH 8.8), 16 mM (NH4)2SO4, 0.01% Tween 20, 200 μM dNTP (Bioron,, Ludwigshafen, Germany), MgCl , 200 nM or 500 nM of each primer for hare (Lep-F/Lep-R) or eukaryotic sequences (18SEU-F/ 18SEU-R), respectively (Table 1), 2.5 mM or 1.5 mM of MgCl2 for hare or eukaryotic DNA, respectively, and 1 U of SuperHot Taq DNA polymerase (Genaxxon Bioscience GmbH, Ulm, Germany). The reactions were performed in a thermal cycler MJ Mini (Bio-Rad Laboratories, Hercules, CA, USA) using the following program: initial denaturation at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 65 °C or 60 °C for hare or eukaryotic DNA, respectively, for 30 s and 72 °C for 45 s; and a final extension at 72 °C for 5 min. The amplified fragments were analysed by electrophoresis in a 2.0% agarose gel containing Gel Red 1x (Biotium, Hayward, CA, USA) for staining and carried out in TAE buffer (40 mM Tris–acetate, 1 mM EDTA) for 60 min at 120 V. The agarose gel was visualised under UV light and a digital image was obtained using a Kodak Digital Science™ DC120 (Rochester, NY, USA). Each extract was amplified at least in duplicate assays. 2.6. Sequencing PCR products To confirm the identity of the PCR products obtained with the Lep-F/Lep-R primers, the expected 127 bp fragments were sequenced. PCR products were purified in Cut&Spin DNA gel extraction columns (GRISP Research Solutions, Porto, Portugal) to remove interfering components. The purified products were sent to a specialised research
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The amplifications by real-time PCR were carried out in 20 μL of total reaction volume containing 2 μL of DNA extract (100 ng to 0.1 pg), 1× of SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and 300 nM of each primer Lep-F/Lep-R (Table 1). The real-time PCR assays were performed on a fluorometric thermal cycler CFX96 Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using the following conditions: 95 °C for 5 min; 45 cycles at 95 °C for 10 s and 65 °C for 30 s, with collection of fluorescence signal at the end of each cycle. Data was collected and processed using the software Bio-Rad CFX Manager 2.0 (Bio-Rad Laboratories, Hercules, CA, USA). For melting curve analysis, PCR products were denatured at 95 °C for 1 min and then annealed at 65 °C for 5 min in order to allow the correct annealing of the DNA duplexes. These two steps were followed by melting curves ranging from 65.0 to 95.0 °C with temperature increments of 0.2 °C every 10 s. The fluorescence data were acquired at the end of each melting temperature. The data collected were processed using the Precision Melt Analysis Software 1.0 (Bio-Rad Laboratories, Hercules, CA, USA). Each reference mixture was analysed in replicates (n = 4) in two independent assays. 3. Results and discussion 3.1. Species-specific PCR This paper reports the specific identification of brown hare based on the design of new primers (Lep-F/Lep-R) targeting the mitochondrial cytb gene of Lepus europaeus. The BLAST results for the expected amplified fragment of 127 bp confirmed 100% specificity for Lepus europaeus, while other Lepus species (L. capensis, L. townsendii, L. yarkandensis, L. oiostolus, L. granatensis, L. brachyurus and L. hainurus) showed a maximum identity from 94 to 96%. The cytb gene was chosen for the present work since it is the most studied gene for phylogeny, being the most targeted gene for molecular identification studies (Teletchea, Maudet, & Hanni, 2005). To verify the specificity of the proposed primers to Lepus species and against other animal species commonly used as food, including game meat, the DNA of wild boar, duck, partridge, quail, rabbit, pheasant, red deer, wild rabbit, cow, chicken, turkey, ostrich, goat and sheep meats was used for PCR amplification. In Fig. 1, the results clearly indicate that Lep-F/Lep-R primers were able to amplify the
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127 bp
Fig. 1. Agarose gel electrophoresis of PCR products targeting Lepus cytb gene. M - 100 bp ladder (Bioron, Ludwigshafen, Germany); lane 1 - wild boar; lane 2 - duck; lane 3 - partridge; lane 4 - hare; lane 5 - quail; lane 6 - rabbit; lane 7 - pheasant; lane 8 – red deer; lane 9 - wild rabbit; lane 10 - cow; lane 11 - chicken; lane 12 - turkey; lane 13 - sheep; lane 14: - goat; lane 15 - ostrich; lane 16 - pork; lane C - negative control.
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Fig. 2. Sequence alignment of mitochondrial cytb entry (AJ534882) with the obtained fragment using primers Lep-F/Lep-R. In italics is represented the sequenced fragment. Underlined is the region of primer annealing. The grey letters represent the mismatching bases.
expected 127 bp of cytb from L. europaeus species. All the extracts were positively amplified to the nuclear 18S rRNA gene for eukaryotic DNA with the universal primers (18SEU-F/18SEU-R), confirming that they contained amplifiable DNA (data not shown). To confirm the homology of the obtained Lepus fragment, the sequenced DNA was aligned with the expected cytb sequence (Fig. 2). The results revealed the occurrence of 7 mismatches, which gives a maximum identity of 95% against the cytb gene of L. europaeus. Performing a new BLAST for the obtained sequence, the results showed a 100% of homology for L. granatensis, which is a species also found in several regions of Portugal and Spain (Alves et al., 2003). Using the Primer-BLAST tool for the proposed primers, the results confirm the complete matching for several cytb entries of L. granatensis, L. europaeus and L. capensis. Many other Lepus species are also probably targeted since they include only one mismatch in one of the primers. This finding supports a closed phylogenetic relationship among several Lepus species (Alves et al., 2003; Ben Slimen et al., 2006; 2008; Pérez-Suarez, Palacios, & Boursot, 1994). The optimised PCR conditions using the binary model mixtures containing known amounts of hare meat in pork meat gave the relative limit of detection of 0.01% of hare meat (Fig. 3). The sensitivity of hare specific PCR assay was determined by amplification of a DNA extract of hare 10-fold serially diluted. The results can be observed in Fig. 4, where the amplification of the lowest level of 10 pg for hare DNA can be seen.
3.2. Real-time PCR Following the successful application of Lep-F/Lep-R primers to species-specific PCR detection, a real-time PCR assay was proposed to identify hare meat. Real-time PCR data collection was achieved
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with the new fluorescent intercalating dye EvaGreen, as a more sensitive and alternative approach than the SYBR Green dye. The results of real-time PCR amplification of hare DNA from 10-fold serially diluted extracts are presented in Fig. 5. The amplification curves (Fig. 5a) indicate the adequacy of the proposed primers for real-time PCR that was confirmed with the respective standard curve (Fig. 5b). The PCR efficiency was very close to the ideal value of 100% that corresponds to a slope of − 3.32, and all data were highly correlated (R 2 > 0.99). The obtained parameters show the appropriateness of the assay for quantitative measurements. The limit of detection was 1 pg, which was improved compared to end-point PCR (10 pg) (Fig. 4). To confirm the specificity of amplified products, a melting curve analysis was performed right after real-time amplification curves. In Fig. 5c, it can be verified that all products have the same melting temperature of 78.2 °C, without showing any evidence of primer dimer or other different fragments. The use of EvaGreen dye contributed to the robust PCR signals obtained as well as strong sharp DNA melt peaks. The relatively high dye concentration in quantitative PCR eliminates dye redistribution problems, making it suitable for fast cycling, high through put and high resolution melting application (Mao, Leung, & Xin, 2007).
4. Conclusions In the present work, two new PCR approaches targeting the mitochondrial cytb gene to identify hare species were proposed. The species-specific PCR assay based on end-point PCR proved to be highly specific and sensitive to hare, reaching a relative level of detection of 0.01% of hare meat and an absolute limit of detection of 10 pg of hare DNA. The second approach based on real-time PCR with the new intercalating EvaGreen dye was also a highly specific technique
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Fig. 3. Agarose gel electrophoresis of PCR products targeting Lepus cytb gene of binary reference mixtures of hare in pork meat. M, 100 bp ladder (Bioron, Ludwigshafen, Germany); lane 1, 100% pork meat; lanes 2–9, different proportions of hare in pork meat; C, negative control.
1 pg
0.5 0.1 0.01%
10 pg
1
1 ng
2.5
10 ng
5
0.1 ng
127 bp 20 10
Fig. 4. Agarose gel electrophoresis of PCR products targeting Lepus cytb gene of different amounts of DNA (lanes 1–7). M, 100 bp ladder (Bioron, Ludwigshafen, Germany); C, negative control.
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a 100 ng 10 ng 1 ng 0.1 ng 10 pg 1 pg 0.1 pg
b
c
Fig. 5. Real-time PCR amplification with EvaGreen intercalating dye targeting the cytb gene of hare. (a) Amplification curves; (b) calibration curve; and (c) melting curves.
for hare identification with a higher sensitivity than end-point PCR (1 pg). The real-time PCR assay proved adequate for quantitative analysis and high throughput hare identification in inspection programs, thus enforcing labelling regulations in the meat industry.
Acknowledgements The authors acknowledge the financial support of U. Porto/Santander Totta “Projectos pluridisciplinares 2010”. This work has also been supported by Fundação para a Ciência e a Tecnologia (FCT) through grant no. PEst-C/EQB/LA0006/2011. The authors are grateful to Prof.
Vasco Cadavez from the Animal Science Department of Polytechnic Institute of Bragança for the supply of samples. References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410. Alves, P. C., Ferrand, N., Suchentrunk, F., & Harris, D. J. (2003). Ancient introgression of Lepus timidus mtDNA into L. granatensis and L. europaeus in the Iberian Peninsula. Molecular Phylogenetics and Evolution, 27, 70–80. Ballin, N. Z., Vogensen, F. K., & Karlsson, A. H. (2009). Species determination – can we detect and quantify meat adulteration? Meat Science, 83, 165–174. Ben Slimen, H., Suchentrunk, Memmi, A., Sert, H., Kryger, U., Alves, P. C., et al. (2006). Evolutionary relationships among hares from North Africa (Lepus sp. or Lepus
C.G. Santos et al. / Meat Science 90 (2012) 836–841 spp.) cape hares (L. capensis) from South Africa, and brown hares (L. europaeus) as inferred from mtDNA PCR-RFLP and allozyme data. Journal of Zoological Systematics and Evolutionary Research, 44, 88–99. Ben Slimen, H., Suchentrunk, F., Stamatis, C., Mamuris, Z., Sert, H., Alves, P. C., et al. (2008). Population genetics of cape and brown hares (Lepus capensis and L. europaeus): A test of Petter's hypothesis of conspecificity. Biochemical Systematics and Ecology, 36, 22–39. Bottero, M. T., & Dalmasso, A. (2010). Animal species identification in food products: Evolution of biomolecular methods. The Veterinary Journal. doi:10.1016/j.tvjl.2010.09.024. Chen, Y., Wu, Y., Wang, J., Xu, B., Zhong, Z., & Xia, J. (2009). Identification of cervidae DNA in feedstuff using a real-time polymerase chain reaction method with the new fluorescence intercalating dye EvaGreen. Journal of AOAC International, 92, 175–180. Cobos, A., Hoz, L., Cambero, M. I., & Ordofiez, J. A. (1995). Chemical and fatty acid composition of meat from spanish wild rabbits and hares. Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 200, 182–185. Colgan, S., O'Brien, L., Maher, M., Shilton, N., McDonnell, K., & Ward, S. (2001). Development of a DNA-based assay for species identification in meat and bone meal. Food Research International, 34, 409–414. Dooley, J. J., Paine, K. E., Garrett, S. D., & Brown, H. M. (2004). Detection of meat species using TaqMan real-time PCR assays. Meat Science, 68, 431–438. European Commission (2004). Regulation (EC) No. 852/2004 of the European Parliament and the Council of 29th of April 2004 on the hygiene of foodstuffs. Official Journal of the European Union, Brussels O.J. L 138/1. Fajardo, V., González, I., Martín, I., Rojas, M., García, T., & Martín, R. (2010). A review of current PCR-based methodologies for the authentication of meats from game animal species. Trends in Food Science and Technology, 21, 408–421. Fajardo, V., González, I., Martín, I., Rojas, M., Hernández, P. E., García, T., et al. (2008). Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures. Meat Science, 79, 289–298. Fettinger, V., Smulders, F. J. M., Lazar, P., Omurtag, I., & Paulsen, P. (2010). Lesions in thighs from hunted Brown Hares (Lepus europaeus) and microflora under vacuum-packaging storage. European Journal of Wildlife Research, 56, 943–947. Girish, P. S., Anjaneyulu, A. S. R., Viswas, K. N., Shivakumar, B. M., Anand, M., Patel, M., et al. (2005). Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene. Meat Science, 70, 107–112. López-Calleja, I., González, I., Fajardo, V., Martín, I., Hernández, P. E., García, T., et al. (2007). Quantitative detection of goats’ milk in sheep's milk by real-time PCR. Food Control, 18, 1466–1473.
841
López-Calleja, I., González, I., Fajardo, V., Martín, I., Hernández, P. E., García, T., et al. (2007). Real-time TaqMan PCR for quantitative detection of cows’ milk in ewes’ milk mixtures. International Dairy Journal, 17, 729–736. Mafra, I., Ferreira, I. M. P. L. V. O., Faria, M. A., & Oliveira, B. P. P. (2004). A novel approach to the quantification of bovine milk in ovine cheeses using a duplex polymerase chain reaction method. Journal of Agricultural and Food Chemistry, 52, 4943–4947. Mafra, I., Ferreira, I. M. P. L. V. O., & Oliveira, M. B. P. P. (2008). Food authentication by PCR-based methods. European Food Research and Technology, 227, 649–665. Mafra, I., Roxo, A., Ferreira, I. M. P. L. V. O., & Oliveira, M. B. P. P. (2007). A duplex polymerase chain reaction for the quantitative detection of cows' milk in goats' milk cheese. International Dairy Journal, 17, 1132–1138. Mafra, I., Silva, S. A., Moreira, E. J. M. O., Ferreira da Silva, C. S., & Oliveira, M. B. P. P. (2008). Comparative study of DNA extraction methods for soybean derived food products. Food Control, 19, 1183–1190. Mao, F., Leung, W. -Y., & Xin, X. (2007). Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology, 7, 76. Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J., et al. (1999). A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science, 51, 143–148. Pérez-Suarez, G., Palacios, F., & Boursot, P. (1994). Speciation and paraphyly in western mediterranean hares (Lepus castroviejoi, L. europaeus, L. granatensis, and L. capensis) revealed by mithocondrial DNA phylogeny. Biochemical Genetics, 32, 423–436. Soares, S., Amaral, J. S., Mafra, I., & Oliveira, M. B. P. P. (2010). Quantitative detection of pork's meat by duplex PCR. Meat Science, 85, 531–536. Somma, M. (2006). Extraction and purification of DNA (Session 4). In M. Querci, M. Jermini, & G. V. Eede (Eds.), Training course on The Analysis of Food Samples for the Presence of Genetically Modified Organisms. European Commission DG-JRC http:// mbg.jrc.ec.europa.eu/capacitybuilding/manuals/Manual%20EN/User%20Manual%20 EN%20full.pdf Teletchea, F., Maudet, C., & Hanni, C. (2005). Food and forensic molecular identification: update and challenges. Trends in Biotechnology, 23, 359–366. Thulin, C. -G., Fang, M., & Averianov, A. O. (2006). Introgression from Lepus europeaus to L. timidus in Russia revealed by mitochondrial single nucleotide polymorphisms and nuclear microsatellites. Hereditas, 143, 68–76. Wang, W., Chen, K., & Xu, C. (2006). DNA quantification using EvaGreen and a real-time PCR instrument. Analytical Biochemistry, 356, 303–305.