Fig.1 (A) Agarose gel electrophoresis of the PCR products amplified from nine mammalian species B, cattle ; P, pig ; K, kangaroo ; G, goat ; S, sheep

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1 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. 45, No. 12, 719 `723 (1998) Identification of Meat Species Based on the Difference of 18 S Ribosomal RNA Genes Takamitsu MATSUNAGA*, Kiyohiro SHIBATA*, Junichi YAMADA*, Yutaka SHINMURA* and Koichi CHIKUNI** *Japan Meat Processors Association, 1-5-6, Ebisu Shibuya-ku, Tokyo ** National Institute of Animal Industry. 2, Ikenodai. Kukizaki-cho. Ibaraki Polymerase Chain Reaction (PCR) was applied to identification of meat and meat products of different species. To distinguish meat from fowl and fish, primers RR-1 (5' -AAACTGCGAA- TGGCTCATTAAATCAGTT-3' ) and RR-6 (5' -ATCGAAAGTTGATAGGGCAGA-3' ) were designed for the regions of 18 S ribosomal RNA gene. The template were 200 ng of genomic DNAs purified from meats of nine mammals, eight birds and two fishes in species. Amplification was carried out 35 cycles of denaturation at 95 Ž for 0.5 min, annealing at 60 Ž for 0.5 min, and extension at 76 Ž for 0.5 min. Amplified products were analyzed on 5% agarose gel electrophoresis. Mammalian DNAs gave 293 by PCR products except kangaroo DNAs from which 317bp fragment was obtained. The PCR products of bird DNA and two fish DNAs gave specific fragments of 254 by and 267 bp, respectively. The sequences of the PCR products from kangaroo, cattle, crocodile, turkey, frog and alaska pollack were determined using the Dye Deoxy Terminator Cycle Sequencing method, for which all PCR products above described are corresponding parts of all 18 S ribosomal RNA gene. The variation in length of PCR products between mammals, birds and fish is due to the difference in 10 loop regions of 18 S ribosomal RNA gene. Based on this difference of 18 S ribosomal RNA gene, several meat products were analyzed by agarose gel electrophoresis. Mixture of pig, chicken and fish in meat products could be identified from each specific fragments by the PCR assay. Each specific fragment from these mixed meat products could be identified by only one series of PCR reaction. (Received Apr. 13, 1998 ; Accepted Aug. 14, 1998)

2 Fig.1 (A) Agarose gel electrophoresis of the PCR products amplified from nine mammalian species B, cattle ; P, pig ; K, kangaroo ; G, goat ; S, sheep ; H, horse ; D, deer ; R, rabbit ; W, whale. M is a molecular marker, ƒõx174/ HincII digest.

Fig.1 (B) Agarose gel electrophoresis of the PCR products amplified from eight avian species C, chicken ; Q, quail ; PI, pigeon ; GF, guinea owl ; DU, duck ; WD, wild duck ; PH, fpheas- ant ; T,

3 Fig.1 (B) Agarose gel electrophoresis of the PCR products amplified from eight avian species C, chicken ; Q, quail ; PI, pigeon ; GF, guinea owl ; DU, duck ; WD, wild duck ; PH, fpheas- ant ; T, turkey. M is a molecular marker, ƒõbx 174/HincII digest. Fig.1 (C) Agarose gel electrophoresis of the PCR products amplified from some animals species and wheat AP, alaska pollack ; AM, acka mackerel ; WH, wheat ; F, frog ; CR, crocodile. M is a molecular marker,ƒõx174/hincii digest.

4 Fig.2 Partial sequences of the 18 S ribosomal RNA genes

2) CHIKUNI, K., OZUTSUMI, K., KOISHIKAWA, T. and KATO, S.: Meat Sci., 27, 119 (1990). 3) WINTERO, A.K., THOMSEN, P.D. and DAVIES, W. Meat Sci., 27, 75 (1990). 4) EBBEHOJ, K.F. and THOMSEN, P.D. : Meat Sci.

5 2) CHIKUNI, K., OZUTSUMI, K., KOISHIKAWA, T. and KATO, S.: Meat Sci., 27, 119 (1990). 3) WINTERO, A.K., THOMSEN, P.D. and DAVIES, W. Meat Sci., 27, 75 (1990). 4) EBBEHOJ, K.F. and THOMSEN, P.D. : Meat Sci., 30, 221 (1991 a). 5) EBBEHOJ, K.F. and THOMSEN, P.D. : Meat Sci., 30, 359 (1991 b). 7) CHIKUNI, K., TABATA, T., KOSUGIYAMA, M., MONMA, M. and SAITO, M.. Meat Sci., 37, 337 (1994). 10) MATSUNAGA, T., CHIKUNI, K., TANABE, R., MUROYA, S., NAKAI, H., SHIBATA, K., YAMADA, J. Fig.3 Agarose gel electrophoresis of the PCR products amplified from meat products 1, Lachsschinken ; 2, Vienna sausage ; 3, Vienna sausage ; 4, Fish sausage. M is a molecular marker, ƒõx174/hincii digest. and SH1NMURA, Y. : Meat Sci.,ˆó ü 11) SAMBROOK, J., FRITSCH, E.F. and MANIATIS, T.: Molecular Cloning, 2nd ed. (Cold Spring Harbour Laboratory Press. New York. USA), (1989). 12) MADEN, B.E. : J. Mol. Biol., 189, 681 (1986). 13) VAN DE PEER, Y., CAERS, A., DE RIJK, P. and DE WACHTER, R. : Nucleic Acids Research., 26, 179 (1998). 14) MCCALLUM, F.S. and MADEN, B.E. : Biochem.J., 232, 725 (1985). 15) RAYNAL, F., MICHOT, B. and BACHELLERIE, J.P. : FEBS Lett., 167, 263 (1984). 16) CHAN, Y.L., GUTELL, R., NOLLER, H.F. and WOOL, I.G. : J. Biol. Chem., 259, 224 (1984). 17) CHIKUMI, K., MINAKA, N. and IKENAGA, H. : J. Yamashina Institute for Ornithology., 28, 1 (1996). 1) BAUR, C., TEIFEL-GREIDING, J. and LEIBHARDT,E.. Arch. Lebensmittelhygiene., 38, 172 (1987).

Table 1 Detection bacterial strains used in this study *1; Methicillin resistant strains(mrsa)

Key words: PCR, ribosomal RNA, bacterial contamination Table 1 Detection bacterial strains used in this study *1; Methicillin resistant strains(mrsa) PCR法 Fig. 1 Agarose gel ribosomal DNA Amplification