Identifying of meat species using

polymerase chain reaction (PCR)

Cite as: AIP Conference Proceedings 1571, 680 (2013); https://doi.org/10.1063/1.4858733
Published Online: 31 December 2013

Chow Ming Foong and Norrakiah Abdullah Sani

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 Identifying Of Meat Species Using Polymerase Chain
Reaction (PCR)
Chow Ming Foong and Norrakiah Abdullah Sani

School of Chemical Sciences and Food Technology, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, 43600,Bangi, Selangor, Malaysia

Abstract. Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy
and intense urban lifestyle, processed food is now one of the main protein sources of one’s diet. Consumers rely on the
food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food
labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality
and safety as compared to before. This study described the meat species identification and detection method using
Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The
objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There
were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of
oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local
raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order
to be applied in food product testing.
Keywords: Species specific, Polymerase Chain Reaction (PCR), meat, identification, authenticity.
PACS: 87.85.M

INTRODUCTION
Food choices normally reflects one’s lifestyle, culture, religion and health concern [1]. Meat has been widely
consumed as an important source of protein intake of a daily diet. Furthermore, due to busy and intense urban
lifestyle, people are lacking of time to prepare food and spending more time in work places. Therefore, processed
meat products has been widely accepted and consumed due to their variety and convenience. Subsequently, food
labeling acts as an important tool to tell the composition of foods.
Recently, the occurrence of incorrect labeling of food been repeatedly reported, be it in milk formula, raw
material like flour, or meat products. Since the occurrence of incorrect labeling of meat in food industry may caused
negative consequences, food authenticity has been a great concern in government authorities, food manufacturer and
consumer [2]. Consumer nowadays made their decision in purchasing food products based on the food labeling.
Therefore, an incorrect or faulty labeling may result in consumer fraud among consumer. Consumer fraud may be
referring to replacement of one meat species with a lower commercial value or nutritional value meat. On the other
hand, faulty labeling may result in food allergies [3] due to undeclared allergen in consumer. Also, consumers who
have religious dietary meat restriction like Muslim [1], Buddhist and Hindu will be defrauded with such replacement
[2]. Lastly, as the increased of food safety issues like outbreak of bovine spongiform encephalopathy (BSE) crisis
[4], there is a need to develop an up to date method to determine adulteration and undeclared meat product in the
market.
There were a variety of methods available to determine and identify the meat species present in food product,
for example the spectroscopy analysis [5], chromatography [6], enzyme-linked immunosorbent assay [7], and DNA-
based technique like polymerase chain reaction (PCR) [2]. PCR been widely used in met species determination since
the 1989 [8]. The DNA based analysis technique using PCR is growing as years gone by. The principles of PCR
involve the amplification of specific length of DNA can be copied and multiplied to provide a sufficient amount of
DNA for confirmation through electrophoresis technique [9]. Therefore, the DNA based technique has the potential
for identification of meat species.
In this study, the identification of meat species using PCR is performed to generate a species specific primer for
met species identification. There are 5 subject of interest of meat species in this study: cattle, sheep, chicken, pig and
horse.

The 2013 UKM FST Postgraduate Colloquium

AIP Conf. Proc. 1571, 680-686 (2014); doi: 10.1063/1.4858733
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 MATERIALS AND METHODS

DNA Extraction from Raw Meat of Animal Species

Meat sample were obtained from the 8 raw muscles of cattle, buffalo, goat, sheep, chicken, duck, pig, and horse.
The raw meat was obtained from a local market in Klang Valley. Samples were transported to the laboratory under
refrigeration of icepack, and they were ready for extraction. Firstly, DNA is extracted from 25 mg of raw meat using
a commercial kit by QIAGEN (DNeasy Blood and Tissue Kit, Darmstadt, Germany). The extracted DNA
concentration was then determined using a MaestroNano Spectrophotometer (MaestroGen, USA). The extracted
DNA sample template was labeled stored separately at -20°C until use.

PCR Oligonucleotide Primer

The nucleotide sequences for 5 different animal species of interest in this study were obtained from literature
[10]. Nucleotides sequences were ordered and purchased from FirstBase (Firstbase, Malaysia). They were stored as
lyophilized aliquots at -20°C until use. The forward and reverse oligonucletides primer sequences for cattle, sheep,
chicken, pig, horse are stated in TABLE (1).

TABLE (1). Oligonucletide primer used in this study.

Target Oligonucleotide Primer Sequence (5’ - 3’) Amplicon Source

F: GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA
Cattle 274 bp
R: CTA GAA AAG TGT AAG ACC CGT AAT ATA AG

F: GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA
Sheep 331 bp
R: CTA TGA ATG CTG TGG CTA TTG TCG CA

F: GAC CTC CCA GCT CCA TCA AAC ACT CAT CTT GAT GAA A Matsunaga et al.10
Chicken 227 bp
R: AAG ATA CAG ATG AAG AAG AAT GAG GCG

F: GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA
Pig 398 bp
R: GCT GAT AGT AGA TTT GTG ATG ACC GTA

Horse F: GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA 439 bp
R: CTC AGA TTC ACT CGA CGA GGG TAG TA

PCR Amplification
PCR amplification was performed using the GreenTaq MasterMix (Promega, USA). A mixture of GreenTaq
MasterMix, nuclease free water (NFW), forward and reserve nucleotides primer, extracted DNA sample template
were prepared in a 0.2 mL centrifuge tube according to the manufacturer’s instruction. On the other hand, a negative
control mixture was prepared by replacing the extracted DNA sample template with NFW. After that, the PCR was
performed using Eppendorf Gradient Thermocycler (Eppendorf, Germany) with 35 cycles of amplification: initial
denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension
72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The final PCR product was kept refrigerated at 4°C
until DNA electrophoresis separation.

DNA Electrophoresis Separation and Image Analyses

After PCR amplification, 8μl of PCR product undergone electrophoresis on a 2% agarose gel (Vivantis, USA) at
100V for 40 minutes in a 1x TAE buffer (40mM Tris-acetate, 1Mm EDTA, pH 8.0, FirstBase, Malaysia). The

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agarose gel was pre-stained with SYBR Safe Gel Stain (Invitrogen, USA). The gel was later viewed and captured
using a gel documentation unit.

RESULTS AND DISCUSSION

FIGURE 1 refers to the agarose gel electrophoresis of PCR product amplified from 8 meats, where LA refers to
la 100kb molecular ladder (New England BioLabs), L1 refers to the extracted cattle DNA template, L2 refers to
extracted buffalo DNA template, L3 refers to extracted goat DNA template, L4 refers to extracted sheep DNA
template, L5 refers to extracted chicken DNA template, L6 refers to extracted duck DNA template, L7 refers to
extracted pig DNA template, L8 refers to extracted horse DNA template, and L9 refers to negative control. While on
the other hand, Figure 1(a) refers to the PCR amplification result with cattle primer, (b) sheep primer, (c) chicken
primer (d) pig primer and (e) horse primer.

Specificity of Oligonucletide Primers for Detection of Raw Meat

Many studies have reported the species-specific PCR methods to determine meat species in food [10-15]. As
mentioned earlier, this study was aimed to develop a new PCR differentiation method with a combination of meat
species. Preventing adulteration and determination of meat species in meat product is an important mission in food
hygiene, food codex, food control and veterinary forensic [16]. In our work, the function of oligonucleotide primers
were tested with running only with targeted extracted DNA template. The results were all positive. To examine the
specificity of each oligonucleotide primer, the primers were examined with other meat species in this study.
Referring to FIGURE 1 (a) which show the direct identification with raw cattle meat, the oligonucleotides primer
proposed by Matsunaga et al. [10] is not species-specific, as the primer amplified with the non-targeted meat
species. A cross-reaction of primers with extracted DNA sample template had occurred at amplicon size 274 bp in
buffalo meat (L2), goat meat (L3), sheep meat (L4), chicken meat (L5) and horse meat (L8) with a high intensity
band in the respective lane. This shows that the primers not only annealed with cattle meat sequences during PCR
amplification, but also annealed with other meat species. The cattle oligonucleotide primer pair may then causes a
false-positive result when tested in meat products that did not contain cattle meat muscle. In a study done by Hsieh
et al., they reported that To avoid cross-reaction or mismatch of primers with targeted raw meat, it’s suggested that
the designing of primers’ at the 3’ end has to be done with great care to avoid nucleotide matching with other meat
species [13].
While on the other hand, it’s found that FIGURE 1 (b) which is the direct identification of raw sheep meat with
proposed primer is not species-specific as cross-reaction occurred at amplicon size 331 bp high intensity band in the
respective lane in buffalo (L2), and goat (L3). The reason of cross-reactivity occurred in buffalo and sheep may due
to some similarity on the long DNA sequence present in DNA sample template. In a research done in India [12] in
year 2004, it’s found that the PCR amplification with their designed primer had cross-reactivity occurred in cattle,
buffalo, goat, chevron and pork. They then proceeded with the species-specific identification with nucleotide
sequencing and analysis. Therefore, nucleotide sequencing can be one of the solutions if cross-reactivity happens. A
study was carried out in Switzerland by Koppel et al. [17] to determine the ability of diagnostic ability, they
concluded that uncertainties were observed for the sheep and goat system. This will lead to the amplification
property of the PCR performed is not sufficient to be characterized and identified.
The PCR amplicon product in FIGURE 1 (c) refers to the proposed chicken primer with raw chicken meat
DNA. From the figure, we can tell that the proposed oligonucleotide primer has cross-reactivity with the extracted
duck DNA sample template (L6) at the amplicon size of 227 bp. Since there were mismatched of primer with
targeted DNA template, other oligonucletides primer can be considered. In s research done by Haunshi et al. [18],
there is a suggestion of oligonucleotides primer which targets chicken meat as a species-specific identification
method. Also, the suggested primer is the study too can be used by 4 different species of jungle fowls and chicken.
On the other hand, another study performed by Mane et al. also suggest PCR assay can be used for amplification of
DNA for available different breeds of chicken to confirm consistency of an amplification result [13].

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(a)

(b)

(c)

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 (d)

(e)
FIGURE 1. Agarose gel electrophoresis of PCR product amplified with 8 types of meats. L1, beef; L2, buffalo; L3, goat, L4,
sheep; L5, chicken; L6, duck; L7, pig; L8, horse; La, 100 kb molecular ladder. (a) PCR amplification with beef primer; (b),
PCR amplification with sheep primer; (c) PCR amplification with chicken primer (d) PCR amplification with pig
primer (e) PCR amplification with horse primer.

In Islam, food containing pork or pig sources are unlawful and non Halal for Muslims to consume [11]. Also, the
world Muslim population has reached 1.8 billion in year 2011 and expected to reach 36% of global population by
year 2025 [19]. Consequently, the consideration of having a pork species specific analysis routine is important in
Malaysia, since the present of pork and lard in food is a serious matter in Islam. From our study, it’s shown that the
proposed pig oligonucleotide primer has occurrence of mismatched with goat (L3) and chicken meat (L5) displayed
in high intensity band in the respective lane, with amplicon size of 398 bp. This shows that the proposed primer is
not pork-specific.
Lastly, FIGURE 1 (e) refers to the DNA amplification of proposed horse primer. From the figure, we can
deduced that there were cross reactivity between horse primer with cattle (L1) and buffalo (L2) with an high
intensity band in respective lane. Occurrence of cross-reactivity can be reduced by having a different annealing
temperature and time. In a study done by Kesman et al. [20], it is reported that the differentiation between horse and
donkey is challenging since they are a closely related species and having a high degree of sequence homology.
However, since horse is consumed in certain countries, but donkey is not so the differentiation is desirable.
According to Martin et al. [21], the demonstration of specificity against a large number of other species is very
important, as when a smaller number of species are used to evaluate the specificity of the assay, there is always a
risk of occurrence of cross-reactivity with the not tested related species, thereby limiting the value of PCR assay.
This is why other than cattle, goat, chicken, other meat species DNA template like buffalo, goat and duck were

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adapted in this study. Subsequently, Ali et al. [19] suggests that amplicon with size less than 150 bp will be a more
suitable to analyses processed food, am a reduction of amplicon size increase the sensitivity.
It also suggested by Mane et al. [13] the authenticity of species-specific PCR method can be done with
confirmation by restriction digestion of amplified DNA fragments with restriction enzyme. The confirmation can be
performed with digestion of PCR products with restriction enzyme. The restriction enzyme selected for the DNA
sequence characterization can then compare with the data available in National Center for Biotechnology
Information(NCBI, www.ncbi.nlm.nih.gov). This method is also adapted in a study performed by Sahilah et al. [15]
to differentiate between pig and wild boat meat using a PCR-Restriction Fragment Length Polymorphism (PCR-
RFLP). Although the alternative technique like PCR-RFLP [2,15], PCR using specific primers has the ability to be a
more useful and cheaper routine analysis for a large samples [21].

CONCLUSION
We need an effective molecular approach to a correct diagnosis in identification of meat species. However, the
proposed oligonucleotide primer in this study were found to be not species specific to cattle, sheep, chicken, pork
and horse. Therefore more study needs to be done with approach of using different primers, either retrieved from
literatures or self-design.

ACKNOWLEDGMENTS
The authors would like to express their greatest gratitude to Universiti Kebangsaan Malaysia for funding this
project with grant INDUSTRI-2011-022and PHI-2011-02.

REFERENCES
1. K. Nakyinsige, Y. B. Che Man and A. Q. Sazili, Meat Science 91 (3), 207 - 214 (2012).
2. A. Pascoal, M. Prado, J. Castro, A. Cepeda and J. Barros-Velazquez, European Food Research and Technology
218, 306 - 312 (2004).
3. H. Hird, R. Goodier and M. Hill, Meat Science 65, 1117 - 1123 (2003).
4. C. Peter, C. Brunen-Nieweler, K. Cammann and T. Borchers, European Food Research and Technology 219,
286 - 293 (2004).
5. H. B. Ding and R. J. Xu, Journal of Food Science 64, 814 - 817 (1999).
6. R. Wissiack, B. de la Calle, G. Bordin and A. R. Rodriguez, Meat Science 64, 427 - 432 (2003).
7. Q. Rao and P. Y.-H. Hsieh, Meat Science 76, 489 - 494 (2007).
8. T. D. Kocher, W. K. Thomas, A. Meyer, S. V. Edwards, S. Paabo, F. X. Villablanca and A. C. Wilson,
Proceedings of the National Academy of Sciences 86, 6196 - 6200 (1989).
9. L. M. Reid, C. P. O'Donnell and G. Downay, Trends in Food Science & Technology 17, 344 - 353 (2006).
10. T. Matsunaga, K. Chikuni, R. Tanabe, S. Muroya, K. Shibata, J. Yamada and Y. Shinmura, Meat Science 51,
143 - 148 (1999).
11. A. A. Aida, Y. B. Che Man, C. M. V. L. Wong, A. R. Raha and R. So, Meat Science 69, 47 - 52 (2005).
12. P. S. Girish, A. S. R. Anjaneyulu, K. N. Viswas, M. Anand, N. Rajkumar, B. M. Shivakumar and S. Bhaskar,
Meat Science 66, 551 - 556 (2004).
13. B. G. Mane, S. K. Mendiratta and A. K. Tiwari, Food Chemistry 116, 806 - 810 (2009).
14. S. B. Ong, M. I. Zuraini, W. G. Jurin, Y. K. Cheak, R. Tunung, L. C. Chai, Y. Haryani, F. M. Ghazali and R.
Son, ASEAN Food Journal 14, 51 - 59 (2007).
15. A. M. Sahilah, W. N. Wan Sakeenah, S. Safiyyah, Y. Norhayati, A. S. Norrakiah, A. Aminah, S. B. Abdul and
A. G. Maaruf, Sains Malaysiana 41, 199 - 204 (2012).
16. A. Doosti, P. G. Dehkordi and E. Rahimi, Journal of Food Science and Technology, doi: 10.1007/s13197-011-
0456-3 (2011).
17. R. Koppel, F. Zimmerli and A. Breitenmoser, European Food Research and Technology 230, 125-133 (2009).
18. S. Haunshi, R. Basumatary, P. S. Girish, S. Doley, R. K. Bardoloi and A. Kumar, Meat Science 83, 454 - 459

685
 (2009).
19. M. E. Ali, M. Kashif, K. Uddin, U. Hashim, S. Mustafa and Y. B. Che Man, Food Analytical Methods, doi:
10.1007/s12161-011-9357-3 (2012).
20. Z. Kesmen, F. Sahin and H. Yetim, Meat Science 77, 649 - 653 (2007).
21. I. Martin, T. Garcia, V. Fajardo, I. Lopez-Calleja, M. Rojas, P. E. Hernandez, I. Gonzalez and R. Martin, Meat
Science 76, 721 - 729 (2007).

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