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 species-specific 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.
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 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 ., 2007
Chikuni . (1994a
) 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 . (1996)
designed multiplex PCR primers based on mitochondrial D-loop DNA sequences and identified cattle, pig, and chicken meats. Subsequently,
Matsunaga . (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 ., 2000
Girish . (2004)
used mitochondrial 12S rRNA gene sequences to identify between cattle and buffalo and between sheep and goat.
Chen . (2012)
analyzed 179 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)]. 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 (
- 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℃ for 10 min a cycle at predenaturation stage. The subsequent denaturation, annealing and extension were run at 95℃, 60℃ and 72℃ for 30 sec, 30 sec and 1 min, respectively and was repeated for 30 cycles. When 5 min extension at 72℃ 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
. The locations of each primer sequence in the target region of the gene were indicated by closed box (
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 (
). Same approaches were applied to the
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 ., 1999
) could not confirm goat DNA when multiplexing was applied (
). We also confirmed that individual species present in meat mixtures were easily identifiable (
), significantly reducing the time and labor required for identifying meats of unknown origin.
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.
Primer pairs (Matsunaga et al., 1999) used for comparative analysis
Primer pairs (Matsunaga et al., 1999) used for comparative analysis
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.
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 3- 5: Goat DNA + goat primer, Lane 6: No DNA + goat primer, Lane 7: Sheep DNA + multiple primers, Lane 8- 10: Goat DNA + multiple primers, Lane 11: No DNA + multiple primers.
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.
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℃, causing the reverse primers to anneal only to species-specific sequences in multiplex PCR. Previously described (
Matsunaga ., 1999
) primers did not confirm goat DNA and this was likely because the mismatch ratio was less than 15％ between the species-specific 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 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.
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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