Download Detection of Meat Origin (Species) Using Polymerase Chain Reaction

Korean J. Food Sci. An.
Vol. 33, No. 6, pp. 696~700(2013)
DOI http://dx.doi.org/10.5851/kosfa.2013.33.6.696
ARTICLE
Detection of Meat Origin (Species) Using Polymerase Chain Reaction
Yong Hyun Park1,2, Md. Rasel Uzzaman3, Jeong-Woon Park2, Sang-Wook Kim3,
Jun Heon Lee1, and Kwan-Suk Kim3*
1
Department of Animal Science & Biotechnology, Chungnam National University, Daejeon 305-764, Korea
2
Institute of Molecular Diagnosis, Genet Bio Corporation, Daejeon 305-500, Korea
3
Department of Animal Science, Chungbuk National University, Cheongju 361-763, Korea
Abstract
A quick and reliable method for identifying meat origin is developed to ensure species origin of livestock products for
consumers. The present study examined the identification of meat sources (duck, chicken, goat, deer, pig, cattle, sheep, and
horse) using PCR by exploiting the mitochondrial 12S rRNA and mitochondrial cytochrome b genes. Species-specific
primers were designed for some or all mitochondrial 12S rRNA nucleotide sequences to identify meat samples from duck,
chicken, goat, and deer. Mitochondrial cytochrome b genes from pig, cattle, sheep, and horse were used to construct speciesspecific primers, which were used to amplify DNA from different meat samples. Primer sets developed in this study were
found to be superior for detecting meat origin when compared to other available methods, for which the discrimination of
meat origin was not equally applicable in some cases. Our new development of species-specific primer sets could be multiplexed in a single PCR reaction to significantly reduce the time and labor required for determining meat samples of
unknown origin from the 8 species. Therefore, the technique developed in this study can be used efficiently to trace the meat
origin in a commercial venture and help consumers to preserve their rights knowing origin of meat products for social,
religious or health consciousness.
Key words: mtDNA 12S rRNA gene, cytochrome b gene, PCR, species-specific primer, multiplexing, meat (species) identification
of meat (species) most consumed in Korea.
Numerous analytical methods for identifying animal
meat species or breeds have been developed based on
protein and DNA analysis (Kesmen et al., 2007). Chikuni
et al. (1994a, b) distinguished between sheep, goat, and
cattle meats using a satellite DNA sequence as well as 8
mammals and 5 birds using the cytochrome b sequence.
Their method involved PCR amplification followed by
restriction digestion, making the procedures for mixed
meats or meat products complicated. Fei et al. (1996)
designed multiplex PCR primers based on mitochondrial
D-loop DNA sequences and identified cattle, pig, and
chicken meats. Subsequently, Matsunaga et al. (1999)
developed a method involving multiplex PCR for the
simultaneous identification of 6 meats (cattle, pig, chicken, sheep, goat, and horse) by using the mitochondrial
cytochrome b gene. Due to its discriminating power in
individual identification, the cytochrome b gene has been
used to distinguish between species (Parson et al., 2000).
Girish et al. (2004) used mitochondrial 12S rRNA gene
sequences to identify between cattle and buffalo and
between sheep and goat. Chen et al. (2012) analyzed 179
Introduction
Developments in the meat industry and higher consumer incomes have led many consumers to be more discriminating in their food choices. The consumption of
meat for protein intake is increasing steadily and consumers who eat meat carefully examine livestock products
for reasons including allergic reactions and religious stipulations, and are concerned about issues such as incorrect
labeling, intentional deceit, cost of meat mixtures, and
improper or illegible products. Therefore, inspection methods for preventing illegal distribution and verifying the
safety of livestock products should be developed. Additionally, the influx of meat from some animals that may
be harmful to humans as well as consumer social needs
should be addressed. Thus, we aimed to develop a reliable molecular technique for tracing the origin and source
*Corresponding author: Kwan-Suk Kim, Department of Animal
Science, Chungbuk National University, Cheongju 361-763,
Korea. Tel: 82-43-261-2547, Fax: 82-43-273-2240, E-mail:
[email protected]
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Detection of Meat Origin using PCR Method
mitochondrial 12S rRNA gene sequences of ten farm animal species to determine the intra-species and speciesspecific variations and could be applied to species identity test, commercial fraud, and wildlife crime. In the
present study, we exploited both the mitochondrial 12S
rRNA and cytochrome b gene sequences to identify mixed
meat (ducks, chickens, goats, deer, pigs, cattle, sheep, and
horses) with PCR.
Materials and Methods
DNA extraction
We obtained meat samples of duck, chicken, goat, deer,
pig, cattle, sheep, and horse from Korea Institute for Animal Products Quality Evaluation. DNA was extracted from
3 to 10 samples of each of the 8 species using a spin-column method (K-3000, Genet Bio Corporation). The purity
and concentration of the isolated DNA were checked with
NanoDrop 1000 Spectrophotometer (Thermo Scientific,
Willington, DE, USA) taking the 260/280 nm. Each DNA
was diluted to have the concentration of 100 ng/µl before
PCR.
Design of species-specific primers
The Species-specific primers were designed using Oligo
6 software (Molecular Biology Insights, Cascade, CO,
USA) for some or all of the mitochondrial 12S rRNA
nucleotide sequences (GenBank accession numbers EU75
5252.1; AY235571.1; GU229278.1; NC_016178.1) from
the NCBI GenBank database to recognize duck, chicken,
goat, and deer, respectively. For the pig, cattle, sheep, and
horse mitochondrial genes (GenBank accession numbers
EF545590.1; JN817351.1; HE577850.1; NC_001640.1,
respectively), species-specific primers were designed to
recognize the entire or part of the mitochondrial cytochrome b gene sequence. The common forward primer
was 12SF (5-GACTAAGAGGAGCTGGTATCARGCAC
AC-3) for duck, chicken, goat, and deer, while the reverse
primers were [duck; (5-GACCCTTGGGTGGAGGCTTG
C-3), chicken; (5-AGCTGGTGTAGATAACATGTGGC-3),
goat; (5-AGCTATAGTGTATCAGCTGCA-3), and deer;
(5-GTAAATAAATTTGAGTGTATTGTGCCTAC-3)]. For
pig, cattle, sheep, and horse, the common forward primer
was MAF (5-GACCTCCCAGCCCCATCAAACATCTC
ATCATGATGAAA-3), while the reverse primers were
[pig; (5-AGCTGATAGTAGATTTGTGATGACCGTA-3);
cattle; (5'-TAGTAGGTGGACTATGGCAATT-3'); sheep;
(5-GCATGAGGATGAGGATTAGTAGGATAGCA-3); and
horse; (5-GAGTGGTATAAAAATGAGAATTAGGGA-3)].
697
PCR primers were designed to generate fragments of different lengths from the 8 species. The sets of primers were
predicted to produce amplicons of approximately 146, 231,
314, 793, 399, 537, 664, and 1047 bp from duck, chicken,
goat, deer, pig, cattle, sheep, and horse, respectively (Fig.
1).
PCR amplification and species-specificity test
DNAs from the different meat samples were amplified
using the species-specific primers. The PCR reaction mixture contained 1 µL (100 ng) of DNA from each species,
0.6 µl (10 µM) forward and reverse primers of any kind
selected from those described in above, and 10 µL PCR
Master Mix (G-7100, HS Prime Taq Premix 2X, GENETBIO, Daejeon, Korea). Sterile distilled water was added
to adjust the final reaction volume to 20 µL. PCR reactions were optimized at 95°C for 10 min a cycle at predenaturation stage. The subsequent denaturation, annealing and extension were run at 95°C, 60°C and 72°C for
30 sec, 30 sec and 1 min, respectively and was repeated
for 30 cycles. When 5 min extension at 72°C PCR was
completed, the products of the PCR were identified using
2% agarose gel electrophoresis at 100 mV for 20 min.
Results and Discussion
The aim of this study was to develop a simple method
for the simultaneous identification of animal species in
meat mixtures. The nucleotide sequences of the mitochondrial genes used to identify meats from duck, chicken, goat, deer, pig, cattle, sheep, and horse are shown in
Fig. 1. The locations of each primer sequence in the target
region of the gene were indicated by closed box (Fig. 1).
To evaluate the specificity of the primers designed in
this study, individual PCR tests were performed. All the
primer pairs were found to be species-specific when specific primer pair applying to multiple species and produced specific bands of 146, 231, 314, 793, 399, 537,
664, and 1,047 bp from duck, chicken, goat, deer, pig, cattle, sheep, and horse DNA, respectively (Fig. 2). Same
approaches were applied to the Table 1 primer set (Matsunaga et al., 1999) and produced species-specific DNA
fragments of 157, 227, 274, 331, 398, and 439 bp from
goat, chicken, cattle, sheep, pig, and horse DNA, respectively (data not shown). We compared the ability of the
two sets of primers mentioned above to identify chicken,
goat, pig, cattle, sheep, and horse species in a multiplexing manner. Our technique clearly identified goat DNA,
whereas a previously described method (Matsunaga et al.,
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Korean J. Food Sci. An., Vol. 33, No. 6 (2013)
Fig. 1. Nucleotide sequences of primers and target regions of the mitochondrial 12S rRNA (A) and cytochrome b (B) genes.
5’-GACTAAGAGGAGCTGGTATCARGCACAC-3’ and 5-GACCTCCCAGCCCCATCAAACATCTCATCATGATGAAA-3 are
two forward primers for the mitochondrial 12S rRNA and cytochrome b genes, respectively. Species-specific primers designed
herein are shown in closed black boxes, respectively.
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Detection of Meat Origin using PCR Method
Table 1. Primer pairs (Matsunaga et al., 1999) used for comparative analysis
Name
Compare-All-F
Compare -Gallus-R
Compare -Capra-R
Comparison -Pig-R
Compare -Cattle-R
Compare -Sheep-R
Compare -Horse-R
Description
Chicken, goat, pig, cattle, sheep and
horse forward primer
Reverse chicken primer
Reverse of goat primer
Reverse pig primer
Reverse cattle primer
Reverse sheep primer
Reverse horse primer
Sequence
5-GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA-3
5-AAGATACAGATGAAGAAGAATGAGGCG-3
5-CTCGACAAATGTGAGTTACAGAGGAA-3
5-GCTGATAGTAGATTTGTGATGACCGTA-3
5-CTAGAAAAGTGTAAGACCCGTAATATAAG-3
5-CTATGAATGCTGTGGCTATTGTCGCA-3
5-CTCAGATTCACTCGACGAGGGTAGTA-3
Fig. 3. Comparative PCR analysis using two different sets of
primers to identify between goat and sheep species.
Lane M: 100 bp DNA marker, Lane 1: Sheep DNA +
sheep primer, Lane 2: No DNA + sheep primer, Lanes 35: Goat DNA + goat primer, Lane 6: No DNA + goat
primer, Lane 7: Sheep DNA + multiple primers, Lane 810: Goat DNA + multiple primers, Lane 11: No DNA
+ multiple primers.
Fig. 2. Individual PCR tests for multiple species. Lane M:
100 bp DNA marker, Lanes 1: Duck DNA, 2: Chicken
DNA, 3: Goat DNA, 4: Pig DNA, 5: Cattle DNA, 6:
Sheep DNA, 7: Deer DNA, 8: Horse DNA.
1999) could not confirm goat DNA when multiplexing
was applied (Fig. 3). We also confirmed that individual
species present in meat mixtures were easily identifiable
(Fig. 4), significantly reducing the time and labor required
for identifying meats of unknown origin.
Multiplex PCR, in which several primers are used together to amplify multiple target regions, is a promising
technique for this purpose. Designing of primers is very
important for multiplex PCR techniques because primer
specificity and melting temperature (Tm) are more critical for this method than for conventional PCR. A high
degree of mismatch (> 15%) decreases the Tm by more
than 15°C, causing the reverse primers to anneal only to
species-specific sequences in multiplex PCR. Previously
described (Matsunaga et al., 1999) primers did not confirm goat DNA and this was likely because the mismatch
ratio was less than 15% between the species-specific
Fig. 4. DNA electrophoresis of PCR products for the animals
species specified in each lane. M: 100 bp DNA marker,
Lane 1: Sheep DNA + horse DNA, Lane 2: Sheep DNA
+ deer DNA, Lane 3: Sheep DNA + cattle DNA, Lane 4:
Sheep DNA + pig DNA, Lane 5: Sheep DNA + goat
DNA, Lane 6: Sheep DNA + chicken DNA, Lane 7:
Sheep DNA + duck DNA.
sheep primer and the template goat DNA sequence; mismatch should be greater than 15% to increase specificity.
We observed that the sheep primers were mismatched
with goat DNA at only two nucleotides, although the 3’end mismatching was fatal for PCR amplification, and
were supposed to produce no sheep band from a goat
template. Therefore it couldn’t bring clear discrimination
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Korean J. Food Sci. An., Vol. 33, No. 6 (2013)
of goat DNA while multiplexing. In our present method,
our primers were designed to amplify target sequences of
8 species with similar efficiencies and resulted in clear
determination of all of them, and can be multiplexed in a
single PCR reaction to significantly reduce the time and
labor to determine the origin of meat samples.
Acknowledgements
This work was supported by a grant entitled “Development of Genetic Improvement Systems in Pigs Using Genomic and Reproductive Technologies” from the Korea
Institute of Planning and Evaluation for Technology of
Food, Agriculture, Forestry and Fisheries.
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(Received 2013.8.9/Revised 2013.10.2/Accepted 2013.10.15)