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    • Abstract: Polimeraz Zincir Reaksiyon (PCR) Yöntemi ile Et Türlerinin Belirlenmesi ... köpek, kedi, s\"¤\"r, koyun, domuz ve keçi etine ait spesifik primerler kullan\"larak Polimeraz Zincir Reaksiyon ...

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Turk. J. Vet. Anim. Sci.
2007; 31(3): 159-163
© TÜB‹TAK Research Article
Identification of Meat Species by Polymerase Chain Reaction (PCR)
Technique*
O. ‹rfan ‹LHAK**, Ali ARSLAN
Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, F›rat University, Elaz›¤ - TURKEY
Received: 21.01.2006
Abstract: The origin of horse, dog, cat, bovine, sheep, porcine, and goat meat was determined by the polymerase chain reaction
(PCR) technique, using species-specific primers. Test mixtures of meat were prepared by adding 5%, 2.5%, 1%, 0.5%, and 0.1%
levels of pork, horse, cat, or dog meat to beef, sheep, and goat meat. Samples taken from those combinations were analyzed by
PCR for species determination. Mitochondrial DNA (mt DNA) fragments of 439, 322, 274, 271, 225, 212, and 157 bp for horse,
dog, cat, bovine, sheep, porcine, and goat meat, respectively, were amplified. PCR was conducted at 30 cycles for mixtures at the
5%, 2.5%, 1%, and 0.5% level, while at 35 cycles for mixtures at the 0.1% level. The results indicated that meat species were
accurately determined in all combinations by PCR. It is concluded that PCR can be useful for fast, easy, and reliable control of
adulterated consumer meat products.
Key Words: Meat species, mt DNA, PCR
Polimeraz Zincir Reaksiyon (PCR) Yöntemi ile Et Türlerinin Belirlenmesi
Özet: Araflt›rmada at, köpek, kedi, s›¤›r, koyun, domuz ve keçi etine ait spesifik primerler kullan›larak Polimeraz Zincir Reaksiyon
(PCR) yöntemi ile etlerde tür tayini yap›ld›. S›¤›r, koyun ve keçi etlerinin her birine % 5, % 2,5, % 1, % 0,5 ve % 0,1 oranlar›nda
ayr› ayr› domuz, at, kedi ve köpek etleri kar›flt›r›larak tür tespiti yap›ld›. Tür tespitinde at, köpek, kedi, s›¤›r, koyun, domuz ve keçiye
ait s›ras›yla 439, 322, 274, 271, 225, 212 ve 157 bp’lik mitokondriyal DNA (mtDNA) parçalar› ço¤alt›ld›. PCR ifllemi; % 5, % 2,5,
% 1 ve % 0,5 oran›ndaki et kar›fl›mlar› için 30, % 0,1 oran›ndaki et kar›fl›mlar› için ise 35 siklusta yap›ld›. Sonuç olarak, PCR yöntemi
ile kolayca, k›sa zamanda ve güvenilir olarak bütün et kar›fl›mlar›nda tür tespiti yap›ld›. Böylece et türlerinin orijini tespit edilerek
halk›n aldat›lmas› engellenece¤i gibi toplumun tüketmedi¤i hayvan etleri di¤er yöntemlere göre daha kolay, h›zl› ve güvenilir bir
flekilde saptanabilir.
Anahtar Sözcükler: Et türleri, mtDNA, PCR
Introduction data about the differences in protein compositions (i.e.
Methods used for identification of species of origin of isoelectrofocusing) (5-7). There is a need for the
raw meat include sensory analysis, anatomical development of a more accurate, fast, and easy-to-use
differences, histological differentiation of the hair that method due to the limitations of the existing methods
may possibly exist in the meat, properties of tissue fat, mentioned above (5).
and level of glycogen in muscle tissue, as well as Developments in molecular biology have facilitated
electrophoresis and DNA hybridization (1-4). Most of identification of plant, bacteria, and animal species with
these methods have been reported to have limitations in high accuracy (8-14). Polymerase chain reaction (PCR),
use due to problems in specificity (i.e. sensory analysis, restriction fragment length polymorphism (RFLP), and
glycogen level, histological differentiation, properties of random amplified polymorphic DNA (RAPD) techniques
tissue fat, and immunological methods), complexity (i.e. have been frequently used for identification of meat
electrophoresis and DNA hybridization), high cost (i.e. species (15-19).
DNA hybridization), and some requirements for baseline
* This article is summarized from the PhD thesis entitled, Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique, by O. ‹. ‹LHAK.
** E-mail: [email protected]
159
Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique
In the present study, the identification of different for 10 min and then the liquid phase was removed. A
meats was determined by PCR, using species-specific 400-ml volume of 70% ethanol was added to the pellet,
primers. In addition, the sensitivity of PCR to identify followed by centrifugation at 11,600 ×g for 5 min for
particular meats in mixtures of meat was determined. washing of the DNA. Finally, ethanol was removed and
the tube containing DNA was held at room temperature
for 30 min for further removal of the residual ethanol via
Materials and Methods evaporation. The pellet, which was the extracted DNA,
Meat samples was diluted with 100 µl of sterile dH2O and used for PCR
Muscle tissue samples from beef, goat, sheep, pig, reaction.
horse, cat, and dog were used. Meat samples were stored Primers
at –20 ± 1 °C until analyzed. PCR primers for the amplification of bovine, sheep,
Test meat mixtures porcine, goat, and horse meat were designed as described
The samples of meat were minced and prepared by Lahiff et al. (21) and Matsunaga et al. (5). Species-
separately by adding 5%, 2.5%, 1%, 0.5%, and 0.1% specific primers (Table) for the detection of dog and cat
(w/w) pork, horse, cat, or dog meat to each of the beef, were designed from sequence information available in the
sheep, and goat meat samples. The mixtures of meat GenBank database (cat: NC_001700,; dog: NC_002008).
were prepared in a total weight of 250 g. Following All primers were obtained from Integrated DNA
mixing, a 2-g portion of each sample was taken Technologies, Inc, (Coralville, IA, USA).
separately from 5 different areas of each test mixture. Polymerase Chain Reaction (PCR)
DNA was extracted from each meat sample and used for The 50-µl reaction mixture was prepared in an
PCR analysis. Eppendorf tube containing 5 µl of 10 × PCR buffer (10
DNA extraction from meats and meat mixtures mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100),
DNA was extracted from meat samples as described 5 µl of 25 mM MgCl2, 250 µM deoxynucleotide
by Koh et al. (20), though with a slight modification. The triphosphate (dNTP), 0.25 µl of Taq DNA polymerase
sample was homogenized using 4 ml of TNES solution (Promega, Madison, WI, USA), 20 pmol of each primer,
(20 mM Tris, (pH 8.0), 150 mM NaCl, and 10 mM EDTA) and 5 µl of target DNA. The thermocycler was
in a 15-ml polypropylene tube. A 750-µl aliquot of the programmed for 30-cycle PCR. PCR was optimized with
resulting homogenate was then transferred into a 1.5-ml different annealing temperatures. The optimal annealing
Eppendorf tube and 10 µl of proteinase K (200 mg/ml) temperature was 58 °C for all primers. Each cycle
and 50 µl of 10% SDS were added. The mixture was included holding at 94 ºC for 45 s, at 58 ºC for 45 s, and
shaken vigorously and kept for 8 h at 58 ºC in a water at 72 ºC for 90 s. For 0.1% meat mixtures, we used 35-
bath. A 250-µl volume of 6 M NaCl was added to the cycle PCR amplification.
resulting mixture and it was centrifuged at 11,600 ×g for Electrophoresis was run on agarose gel (1.5%) at
5 min. A 500-µl portion of the aquatic phase of the 100 V for 2 h on a 15-µl portion of the amplified DNA
sample was then transferred into a separate Eppendorf fragments. The resulting gel was stained with ethidium
tube and 300 µl of a phenol-chloroform-isoamyl alcohol bromide (0.5 µg/ml), visualized using a UV
(25:24:1) mixture was added, followed by vigorous transilluminator, and photographed with a Polaroid 322
shaking and centrifugation at 11,600 ×g for 5 camera and T667 film. The experiments were conducted
min. A 400-µl portion of the upper layer was then in triplicate.
transferred into another tube and 300 µl of chloroform
was added, followed by mixing and centrifugation. A
300-µl portion of the upper phase was then taken and Results
400 µl of absolute ethanol at –20 ºC and 40 µl of sodium Mitochondrial DNA (mt DNA) fragments of 439, 322,
acetate were added prior to vortexing and storing 274, 271, 225, 212, and 157 bp of horse, dog, cat,
the sample at –20 ºC for 8 h for precipitation of DNA. bovine, sheep, porcine, and goat meat, respectively, were
The resulting mixture was then centrifuged at 11,600 ×g amplified (Figure 1). None of the primer pairs used cross-
160
O. ‹. ‹LHAK, A. ARSLAN
Table. PCR oligonucleotide primers.
Position Accession number
Bovine 5’- GCCATATACTCTCCTTGGTGACA- 3’ 8107/8127 J01394
5’- GTAGGCTTGGGAATAGTACGA- 3’ 8377/8357
Sheep 5’- TTAAAGACTGAGAGCATGATA- 3’ 71/91 AF039171
5’- ATGAAAGAGGCAAATAGATTTTCG- 3’ 295/272
Porcine 5’- GCCTAAATCTCCCCTCAATGGTA- 3’ 93/115 AF039170
5’- ATGAAAGAGGCAAATAGATTTTCG- 3’ 304/281
Cat 5’- CATGCCTATCGAAACCTAACATAA- 3’ 11101/11124 NC_001700
5’- AAAGAAGCTGCAGGAGAGTGAGT- 3’ 11373/11351
Dog 5’- GATGTGATCCGAGAAGGCACA- 3’ 8821/8841 NC_002008
5’- TTGTAATGAATAAGGCTTGAAG- 3’ 9142/9121
Reference
Goat
5’- GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA- 3’ (Matsunaga et al., 1998)
5’- CTCGACAAATGTGAGTTACAGAGGGA- 3’
Horse
5’- GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA- 3’ (Matsunaga et al., 1998)
5’- CTCAGATTCACTCGACGAGGGTAGTA- 3’
reacted with DNA of other species. Test mixtures of meat
at 5%, 2.5%, 1%, and 0.5% levels were identified after
an amplification of 30 cycles, while identification failed
for 0.1% mixtures (Figure 2). However, 0.1% mixtures
were identified with 35 amplification cycles (Figure 3).
Discussion
Species identification of meat and meat products is
important because of health, ethical, and economic
reasons. Wintero et al. (22) compared immunodiffusion,
immunoelectrophoresis, isoelectric focusing, and DNA-
hybridization for determining species of meat. They
concluded that DNA hybridization was more reliable and
sensitive than other methods, though it was complicated
and time-consuming. Similarly, the high cost and
complexity associated with this technique have been
reported by other researchers (19,20).
Meyer et al. (7) detected 0.5% pork in beef using the
Figure 1. Agarose gel analysis of PCR product amplified with species- duplex PCR technique. Their results revealed that PCR
specific primers. was the method of choice for identifying meat species in
M: molecular marker (100 bp); 1: horse meat; 2: dog meat; muscle foods. Meyer et al. (19) detected 0.01% soy
3: cat meat; 4: beef; 5: lamb; 6: pork; 7: goat meat.
161
Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique
Figure 3. Agarose gel analysis of PCR products from meat mixtures at
0.1% level (35 PCR cycles)
M: molecular marker (100 bp); 1: 0.1% pork in beef; 2:
0.1% pork in lamb; 3: 0.1% pork in goat meat; 4: 0.1% cat
Figure 2. Agarose gel analysis of PCR products from mixtures of beef- meat in beef; 5: 0.1% cat meat in lamb; 6: 0.1% cat meat in
horse meat with horse-specific primer (30 PCR cycles) goat meat; 7: 0.1% dog meat in beef; 8: 0.1% dog meat in
M: molecular marker (100 bp); 1: 100% beef (beef-specific lamb; 9: 0.1% dog meat in goat meat; 10: 0.1% horse meat
primer is used to indicate the presence of beef); 2: 100%
in beef; 11: 0.1% horse meat in lamb; 12: 0.1% horse meat
horse meat (positive control): 3: 5% horse meat in beef; 4:
in goat meat.
2.5% horse meat in beef; 5: 1% horse meat in beef; 6:
0.5% horse meat in beef; 7: 0.1% horse meat in beef; 8:
100% beef (negative control: horse-specific primer is used to
indicate the absence of horse meat).
protein in processed meat products using the nested-PCR samples may result in positive results for a violation due
technique. Partis et al. (23) detected 1% pork in beef to its high sensitivity (3,6), even though contamination
using RFLP, whereas Hopwood et al. (17) detected 1% was unintentional and at a very low level. Therefore,
chicken in lamb using PCR. precaution should be exercised when interpreting the
Results of the present study supported the findings results of species identification by PCR and analysis of
published by Meyer et al. (6,7), Hopwood et al. (17), and multiple samples should be taken from each lot for an
Partis et al. (23), who reported that PCR could be used objective evaluation.
for identification of meat mixes at 1% and 0.5% levels. These results might be useful for effective control of
Our results suggested that the number of PCR cycles used adulterated consumer meat products and violations of
for amplification played an essential role in identification labeling requirements for meat products. PCR species
of meat in mixes < 0.5%. Therefore, in cases where a determination can also be used to monitor ruminant feeds
very low level of meat is suspected of being mixed into for any beef tissue, which has been banned in many
the main meat batch, the meat batch should be countries in an effort to control the spread of bovine
homogenized before sampling, multiple samples should spongiform encephalopathy.
be taken, and the number of PCR amplification cycles
should be increased (i.e. 35).
Acknowledgment
In meat plants processing more than one species of
meat, it may be inevitable that one species of meat may We thank Dr. M. Calıcıo¤lu for assistance with writing
be contaminated with another during meat operations, this manuscript. We also thank the Scientific Project Fund
such as cutting and grinding via knives, grinders, of Fırat University for supporting this work (Project No:
choppers, and cutting boards. PCR analysis of such 691).
162
O. ‹. ‹LHAK, A. ARSLAN
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