A quick and simple method for the identi cation of meat species and meat products by PCR assay

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1 Meat Science 51 (1999) 143±148 A quick and simple method for the identi cation of meat species and meat products by PCR assay T. Matsunaga a, K. Chikuni b *, R. Tanabe b, S. Muroya b, K. Shibata a, J. Yamada a, Y. Shinmura a a Japan Meat Processors Association, Ebisu Shibuya-ku, Tokyo 150, Japan b National Institute of Animal Industry, Tsukuba Norindanchi, PO Box 5, Ibaraki 305, Japan Received 28 August 1997; received in revised form 30 June 1998; accepted 2 July 1998 Abstract The polymerase chain reaction (PCR) was applied to identify six meats (cattle, pig, chicken, sheep, goat and horse) as raw materials for products. By mixing seven primers in appropriate ratios, species-speci c DNA fragments could be identi ed by only one multiplex PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial cytochrome b gene, and reverse primers on species-speci c DNA sequences for each species. PCR primers were designed to give di erent length fragments from the six meats. The products showed species-speci c DNA fragments of 157, 227, 274, 331, 398 and 439 bp from goat, chicken, cattle, sheep, pig and horse meats, respectively. Identi cation is possible by electrophoresis of PCR products. Cattle, pig, chicken, sheep and goat fragments were ampli ed from cooked meat heated at 100 or 120 C for 30 min, but horse DNA fragments could not be detected from the 120 C sample. Detection limits of the DNA samples were 0.25 ng for all meats. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: PCR; Meat species identi cation 1. Introduction Accurate analytical methods are indispensable for the labeling of meat products, and that requires simple and fast procedures. DNA hybridization (Baur et al., 1987; Chikuni et al., 1990; Winterù et al., 1990; Ebbehùj and Thomsen, 1991a, b) and PCR methods (Chikuni et al., 1994a, b; Fei et al., 1996) have been used for identi cation of meats and meat products. DNA hybridization methods are complicated and generally inadequate, but PCR easily ampli es target regions of template DNA in a much shorter time (Saiki et al., 1985), and thus is suitable for meat identi cation. Chikuni et al. (1994a, b) distinguished sheep, goat and cattle meats using a satellite DNA sequence and eight mammals and ve birds using the cytochrome b sequence. That method consisted of PCR ampli cation followed by restriction digestions, thus the procedures for mixed meats or meat products were complicated. Fei et al. (1996) designed multiplex PCR primers based on mitochondrial D-loop * Corresponding author. rc.go.jp DNA sequences and identi ed cattle, pig and chicken meats. In the present study, the authors developed a simple method using multiplex PCR for simultaneous identi cation of six meats. 2. Methods 2.1. DNA extraction All meat samples were obtained from commercial sources. DNA was prepared from cattle, pig, chicken, sheep, goat and horse meats as described by Sambrook et al. (1989). DNA/RNA mixtures were extracted in 20 vol of 100 mm Tris±HCl, (ph 9.0) containing 100 mm NaCl, 5 mm EDTA and 1% SDS, for 30 min at the room temperature. The DNA/RNA solutions were extracted with an equal volume of phenol/chloroform/ isoamyl-alcohol (25:24:1), and then with an equal volume of chloroform. RNA in the DNA/RNA solution was degraded by 100 mg ml 1 Ribonuclease A (Sigma) for 1 h at 37 C. DNA was extracted twice with an equal volume of phenol/chloroform/isoamyl-alcohol /98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved PII: S (98)

2 Fig. 1. Nucleotide sequences of the primers and target region on cytochrome b gene. Open boxes indicate the common forward primer SIM and complementary sequences of species-speci c reverse primer. Dots and closed boxes indicate identical and di erent nucleotides to the primer sequences, respectively. 144 T. Matsunaga et al./meat Science 51 (1999) 143±148

3 T. Matsunaga et al./meat Science 51 (1999) 143± and once with an equal volume of chloroform. DNA concentrated by ethanol precipitation was dissolved in 10 mm Tris±HCl, (ph 7.4) 0.1 mm EDTA for use as the PCR template. DNA concentration was determined based on absorbance at 260 nm. Each piece of 25 g from cattle, pig, chicken, sheep, goat and horse meats was heated for 30 min at 100 or 120 C. The DNA/RNA mixture was extracted from 500 mg of heated meat with 10ml of 100 mm Tris±HCl, (ph 9.0) containing 1% SDS and 5 mm DTT for 5 min at 95 C. The DNA solution was puri ed by phenol/chloroform/isoamylalcohol extraction Polymerase chain reaction (PCR) PCR ampli cation was conducted in 50 ml of 10 mm Tris±HCl, (ph 8.3) containing 50 mm KCl, 1.5 mm MgCl 2, 200 mm dntp mix, primer mix (4±60 pmol each), 250 ng template DNA and 1.25 unit Taq DNA polymerase (Perkin±Elmer). Oligonucleotide primers were the common forward primer SIM (5 0 -GACCTC- CCAGCTCCATCAAACATCTCATCTTGATGAAA- 3 0 ) and reverse primers [goat primer G (5 0 -CTCGA- CAAATGTGAGTTACAGAGGGA-3 0 ), chicken primer C (5 0 -AAGATACAGATGAAGAAGAATGAGGCG- 3 0 ), cattle primer B (5 0 -CTAGAAAAGTGTAAGA- CCCGTAATATAAG-3 0 ), sheep primer S (5 0 -CTATG- AATGCTGTGGCTATTGTCGCA-3 0 ), pig primer P (5 0 -GCTGATAGTAGATTTGTGATGACCGTA-3 0 ), and horse primer H (5 0 -CTCAGATTCACTCGACG- AGGGTAGTA-3 0 )] (Fig. 1) that were designed from published sequences of cattle, pig, chicken, sheep, goat and horse mitochondial cytochrome b genes (Anderson et al., 1982; Irwin et al., 1991; Desjardins and Morais, 1990). These primers were mixed in the ratio of 1:0.2:3:0.6:3:0.6:2 for SIM:G:C:B:S:P:H, and used together for the multiplex PCRs of this study (the ratio 1 means 20 pmol primer/50 ml PCR solution). Thirty- ve cycles of ampli cation were run using a GeneAmp PCR System 2400 (Perkin±Elmer) as follows: denaturation at 94 C for 0.5 min, annealing at 60 C for 0.5 min, and extension at 72 C for 0.5 min. Following ampli cation, 8 ml PCR solution was electrophoresed on 4% NuSieve GTG agarose gel (FMC) for 30 min at 100 V in TAE bu er (40 mm Tris±acetate, 1 mm EDTA, ph 8.0) and then stained with ethidium bromide (0.5 mg ml 1 ) for 1 h. on 38 bp sequence. Because each 1% mismatching of bases in a double-stranded (ds) DNA reduces melting temperature (Tm) by 1±1.5 C (Sambrook et al., 1989), the mismatches of SIM would decrease the Tm of primer-template dsdna about 10±15 C. Thus, SIM was designed as longer than species-speci c primers that were 26±29 nucleotides long. The reverse primers were designed on species-speci c regions. The primers were expected to amplify target sequences at the same e ciency in multiplex PCR. Fig. 2 shows 4% agarose gel electrophoresis of PCR products ampli ed from the six species. PCR products from goat, chicken, cattle, sheep, pig and horse DNAs were single DNA fragments of 157, 227, 274, 331, 398 and 439 bp, respectively. The six meats could thus be identi ed based on the length of PCR products with no cross reaction Identi cation of cooked meat Cooked samples were obtained from the six meats heated for 30 min at 100 or 120 C, and DNA extracted from 500 mg of the meats was used as a template for PCR. The PCR products were analyzed by 4% agarose gel electophoresis. Fig. 3 shows the species-speci c DNA fragments to have been ampli ed from the cooked meat heated at 100 and 120 C except for horse meat cooked at 120 C. The cattle fragment was ampli- ed from 120 C sample, but the signal was weaker than the other species. 3. Results 3.1. Meat identi cation by species-speci c primers Fig. 1 shows primer regions on cytochrome b sequences of the six species. The common forward primer SIM mismatches with the six species at 3±5 bases Fig. 2. Agarose gel electrophoresis of PCR product ampli ed from the six meats. G, goat; C, chicken; B, cattle; S, sheep; P, pig; H, horse. M is a molecular marker, f174/hincdigest.

4 146 T. Matsunaga et al./meat Science 51 (1999) 143±148 Fig. 3. Agarose gel electrophoresis of PCR products ampli ed from cooked meat. G, goat; C, chicken; B, cattle; S, sheep; P, pig; H, horse. Lane (±) is a PCR product ampli ed from a reaction solution without a template DNA. M is a molecular marker, f174/hincdigest Semi-quanti cation of mixed DNA DNAs extracted from cattle and pig meats were mixed for use as templates in the ratios of 88:12, 75:25, 50:50, 25:75 and 12:88. Fig. 4 shows the bands of cattle and pig-speci c fragments of 274 and 398 bp, along with the relationships between template DNA amounts and band intensity. In lane 4 (50:50), two bands from cattle and pig DNAs indicated similar amounts of PCR products. When pig DNA increased, from lanes 2±6, the band of 398 bp fragment became intense and that of 274 bp fragment faint Detection limits of DNA samples Fig. 5 shows the results of PCR ampli cation from mixed DNA templates of 25, 2.5, 0.25, and ng each. Low molecular bands in all lanes in many cases were probably primer±dimers produced from seven primers during PCR. Lanes 1±3 show six bands corresponding to the six species, thus the detection limits was 0.25 ng for all meat species. 4. Discussion Fig. 4. Agarose gel electrophoresis of PCR products ampli ed from cattle and pig DNA mixture. Lanes 1,(100:0); 2,(88:12); 3,(75:25); 4,(50:50); 5,(25:75); 6,(12:88); 7,(0:100). M is molecular marker, f174/hincdigest. The aim of this study was to develop a simple method for simultaneous identi cation of multiple meat species. Multiplex PCR, in which many primers are used together for ampli cation of multiple target regions, is a hopeful technique for this purpose. The design of primers is very important on multiplex PCR techniques, because primer speci city and Tm are more critical than conventional PCRs. Ratios of mismatching in this study were more than 15% between a species-speci c primer and the other species sequences except a pair of sheep primer and goat template. The mismatches more than 15% decrease Tm more than 15 C, and that make reverse primers anneal only to the species-speci c sequence in multiplex PCR. The sheep primer S mismatched with goat DNA at only two nucleotides, however, 3 0 end mismatching was fatal for PCR ampli cation and resulted in no sheep band from a goat

5 T. Matsunaga et al./meat Science 51 (1999) 143± Fig. 5. Agarose gel electrophoresis of PCR products ampli ed from , 0.025, 0.25, 2.5 and 25 ng from DNA of the six meats. Lanes 1, 2, 3, 4 and 5 are 25, 2.5, 0.25, and ng of six meats DNA, respectively. Lane 6 is a PCR product ampli ed from the reaction solution without template DNA. M is molecular marker, f174/ Hincdigest. template (Fig. 2). The other primers were also designed to mismatch with di erent species at 3 0 end or next nucleotides (Fig. 1). Primer speci city in the entire DNA of a target species was examined by conventional PCR using a pair of SIM and an each species-speci c primer. The size of PCR products was as expected with no additional fragment from a target species (data not shown). This result showed that the species-speci c primers ampli ed only one size fragment from a target species. Primer speci city to the other species was examined by multiplex PCR using the same primer mixture in the method. Fig. 2 showed that multiplex PCR resulted in a single band of target size from one meat species and no fragment produced by non-speci c ampli cation. The primers were designed to amplify target sequences of the six species at similar e ciency. Fig. 6 shows, however, multiplex PCRs using equal amount mixture of the primers did not result in equal signals from the six species (lanes 1,2). In general, quantitative PCR is di cult because of unequal e ciency of ampli cation. Ampli cation e ciency is a ected by the di erence of primer sequences. The common primer SIM was designed to be shared by the six species, therefore, ampli cation e ciency of PCR was a ected by only Fig. 6. Agarose gel electrophoresis of PCR products ampli ed from DNA of the six meats. Lanes 1, 12.5 ng; 2, 25 ng; 3, 12.5 ng; 4, 25 ng. Lanes 1 and 2 are the products by mixed primers of the equal concentration. Lanes 3 and 4 are of the changed concentration. M is molecular marker, f174/hincdigest. reverse primers. A little di erence of Tm among the reverse primers would a ect the e ciency. In order to control the e ciency, the ratio of the primers was changed according to the results of preliminary experiments. Lanes 3 and 4 in Fig. 6 showed that all bands from the six species were detectable by using an appropriate ratio of primer mixture and Fig. 4 showed a possibility of semi-quantitative analysis for beef and pork mixture. However, quantitative ampli cation was found to be unsuccessful as for the other species. Fig. 5 showed di erent signal intensity from the same concentration of DNA of the six species. Multiplex PCRs detected meat species prepared at high temperature (Fig. 3). Cooked meat DNAs (except horse) were ampli ed from all samples heated at all temperatures, but no horse DNA fragment could be detected at 120 C. Because 439 bp horse DNA was the longest fragment, targeted horse DNA fragment would be a ected more by heat than other DNA fragments at high temperature. The primer for horse should thus be designed to amplify a shorter fragment. PCR is quite useful for routine analysis of meat species identi cation, being quick and sensitive. By the present method, the six meats could all be identi ed at the same time more easily and sensitive than usual methods. The analytical conditions must be improved for quantitative di erentiation.

6 148 T. Matsunaga et al./meat Science 51 (1999) 143±148 References Anderson, S., de Bruijin, M.H.L., Coulson, A.R., Eperon, I.C., Sanger, F., Young, I.G., Complete sequence of cattle mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. Journal of Molecular Biology 156, 683±717. Baur, C., Teifel-Greiding, J., Leibhardt, E., Spezi zierung hitzedenaturierter Fleischproben durch DNA-Analyse. Archiv fur Lebensmittelhygiene 38, 172±174. Chikuni, K., Ozutsumi, K., Koishikawa, T., Kato, S., Species identi cation of cooked meats by DNA hybridization assay. Meat Science 27, 119±128. Chikuni, K., Tabata, T., Kosugiyama, M., Monma, M., Saito, M., 1994a. Polymerase chain reaction assay for detection of sheep and goat meats. Meat Science 37, 337±345. Chikuni, K., Tabata, T., Saito, M., Monma, M., 1994b. Sequencing of mitochondrial cytochrome b genes for the identi cation of meat species. Animal Science and Technology (Jpn) 65, 571±579. Desjardins, P., Morais, R., Sequence and gene organization of the chicken mitochondrial genome: A novel gene order in higher vertebrates. Journal of Molecular Biology 212, 599±634. Ebbehùj, K.F., Thomsen, P.D., 1991a. Species di erentiation of heated meat products by DNA hybridization. Meat Science 30, 221±234. Ebbehùj, K.F., Thomsen, P.D., 1991b. Di erentiation of closely related species by DNA hybridization. Meat Science 30, 359±366. Fei, S., Okayama, T., Yamanoue, M., Nishikawa, I., Mannen, H., Tsuji, S., Species identi cation of meats and meat products by PCR. Animal Science and Technology, (Jpn) 67, 900±905. Irwin, D.M., Kocher, T.D., Wilson, A.C., Evolution of the cytochrome b gene of mammals. Journal of Molecular Evolution 32, 128±144. Saiki, R.K., Schart, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., Arnheim, N., Enzymatic ampli cation of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350±1354. Sambrook, J., Fritsch, E.F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbour Laboratory Press, New York. Winterù, A.K., Thomsen, P.D., Davies, W., A comparison of DNA-hybridization, immunodi usion, countercurrent immunoelectrophoresis and isoelectric focusing for detecting the admixture of pork to beef. Meat Science 27, 75±85.

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