82 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.,, 2, 20 (,**1) 22 * * Specific Primer for Detection of Wheat Masataka Uchino, Namiko Masubuchi, Y

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1 82 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.,, 2,20 (,**1) 22 * * Specific Primer for Detection of Wheat Masataka Uchino, Namiko Masubuchi, Yukiko Kurosawa, Tomohiro Noguchi* and Katsumi Takano Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, Sakuragaoka +++, Setagaya-ku, Tokyo +/02/*, * Food Processing Center, Tokyo University of Agriculture, Sakuragaoka +++, Setagaya-ku, Tokyo +/02/*, Because wheat contains a protein that can trigger allergies, the presence of wheat must be indicated on food products in Japan. One method for detection of wheat is by PCR. However, in the general detection method, only one primer set is used. In this study, we developed a new primer set for detection of wheat to protect against false results. Two wheat primer sets, + and,, were constructed. The PCR product using set + showed a band around the same molecular weight not only as wheat and rye-wheat containing an allergen compound, but also rye. Two amplified DNA sequences specific to wheat and rye-wheat, using primer set,,2** bp and /** bp, were cloned and the sequences were determined. Two STS primer sets, K2** and K/**, were reconstructed based on these sequences. PCR conducted using these sets showed specific bands, 2** bp, /** bp and./* bp. These were useful for testing of food products containing wheat. In addition, our results showed that the K/** set was superior to the K2** set. (Received Jun.,0,,**0 ; Accepted Oct.,0,,**0) Keywords : Wheat, Detection, Primer, PCR : PCR,**+ /,* http : // + +/ 0/* / ELISA PCR PCR +2 DNA 3 PCR + +/02/*, +++ * +/02/*, +++ Corresponding author muchino@nodai.ac.jp + + Aspergillus kawachii, Saccharomyces cerevisiae, Saccharomyces pombe, DNA PCR DNA Gen Elute Plant Genomic DNA Kit SDS +* - ISOPLANT DNA

2 23 : O.D.,0* nm/o.d.,2* nm +./ DNA DNA PCR +2S rdna +, -,*.*, + :UNI,f, /TGAACSTCGTGTTCGTC- : VIEr, /AGCTTCATGTAGGCGA-, : WHEf, /-AGTTCCGAAGAGA- TCAGACAA-- : GBSSf, /-ATCACATGGC- AAGCATCACA-- UNI,f, GBSSf WHEf VIEr Bacillus vietnemensis DNA +** ngl + l, +*PCR / l,,./ mm dntp. l,,*pml *./ l, Ex Taq DNA / Ul, *., l, -2.2 l /* l PCR 3. / 3. *./ /+ +.* 1, +./ -* 1, 1 PCR Genamp 31** PCR., PCR LaboPass Gel pgem-t Easy Vector System+ E. coli JM +*3 Micropulser SDS ++ M+- Big Dye ver. -.+ Geneamp 31** DNA ABI -+** Avant, DNA BLAST FASTA DDBJ : http : // +, / STS PCR Clustal W +. GC DNA STS STS PCR K2** /* K/** // PCR 0 PCR DNA Gen Elute Plant Genomic DNA Kit / PCR + + DNA PCR Fig. +.+* bp, A. kawachii 1** bp /** bp, Fig., A. kawachii 2** bp /** bp +,,,, +,,, PCR PCR

84 /.,,**1, 24 Fig. + Electrophoresis of amplified DNA using WHEf-GBSSf primer set Abbreviations : +, Wheat ;,, Non-glutinous rice ; -, Barley ;.

, Soybean ; M, Molecular weight marker. Fig., Electrophoresis of amplified DNA using UNI,f- VIEr primer set Abbreviations : +, Wheat ;,, Non-glutinous rice ; -, Barley ;.

DNA database K2** K/** +* GC STS 2** bp /, - K2**r /-CGTGACAGAAGGTGATGAGGCT-- /** bp K /**f /-TCGTGTTCGTCATCATCGTC-- K/**r /-CTTCATGTAGGCGAAACCGA-- DNA PCR Fig. - K2** Fig.

3 84 /.,,**1, 24 Fig. + Electrophoresis of amplified DNA using WHEf-GBSSf primer set Abbreviations : +, Wheat ;,, Non-glutinous rice ; -, Barley ;., Foxtail millet ; /, Proso millet ; 0, Japanese barnyard millet ; 1, Sorghum ; 2, Rye-wheat ; 3, Rye ; +*, A. kawachii ; ++, S. cerevisiae ; +,, S. pombe ; +-, Rat ; +., Soybean ; M, Molecular weight marker. Fig., Electrophoresis of amplified DNA using UNI,f- VIEr primer set Abbreviations : +, Wheat ;,, Non-glutinous rice ; -, Barley ;., Foxtail millet ; /, Proso millet ; 0, Japanese barnyard millet ; 1, Sorghum ; 2, Rye-wheat ; 3, Rye ; +*, A. kawachii ; ++, S. cerevisiae ; +,, S. pombe ; +-, Rat ; +., Soybean ; M, Molecular weight marker.,, 2** bp /** bp K2** K/** Table + FASTA BLAST, DNA query vs. DNA database K2** K/** +* GC STS 2** bp /, - K2**r /-CGTGACAGAAGGTGATGAGGCT-- /** bp K /**f /-TCGTGTTCGTCATCATCGTC-- K/**r /-CTTCATGTAGGCGAAACCGA-- DNA PCR Fig. - K2** Fig. - Wheat-specific bands amplified with STS primer Abbreviations : +, Wheat ;,, Non-glutinous rice ; -, Barley ;., Foxtail millet ; /, Proso millet ; 0, Japanese barnyard millet ; 1, Sorghum ; 2, Rye ; 3, Rye-wheat +*, A. kawachii ; ++, S. cerevisiae ; +,, S. pombe ; +-, Rat ; +., Soybean ; M, Molecular weight marker.

25 : 85 Table + Sequences of two wheat-specific bands amplified with UNI,f-VIEr primer sets GGGCTATCGTCTTGGTGCCTAGCAAGAAGTGTCTGGAATACTCATGGCA CACGATTAGTCTCGCAAATGCGTTGTCATCAATCACCAAAACACTTAGG

4 25 : 85 Table + Sequences of two wheat-specific bands amplified with UNI,f-VIEr primer sets GGGCTATCGTCTTGGTGCCTAGCAAGAAGTGTCTGGAATACTCATGGCA CACGATTAGTCTCGCAAATGCGTTGTCATCAATCACCAAAACACTTAGG GATAAATATGTCCTTACAATCTCCCCCTTTTTGGTGGATTGATGACAAAG CGGGATTTGCACAAGGAAAATATACTATTAAGTAAAAGGCAAACCCCTC CCTCTCTATAATATAGATGGGCTCCCCCTAAATGTGTGCCACCTAGATG AGCACGAATACTAGGAACAGAGCTCCCCCTATATTATATAGACAAGGCA AATTTATCACTAGACAAGATAGTACAGACATAAGATAACTAAGATAGAT AGAGTGCATATGTCTTACACCATATGGAGGATAGGTCTCGAAGGCACAA ACCGAACAAAAGCAAACGAGGCAAACAAGGTTCAAACGACAAAGCAA ACAAACACACCAAGCACGACACAACGCAAATCCCTACACTCTCTCCCC CTTTGGCATCGAGACGCCAAAAAGGCAGAGAGGACACCCTACACACAC GGGGTGGCTCAGGCAGAGAAGTCGTCCCACTGCTCCTCGTGCTCCTCG TCAGACTCAGCAGGCGGGGATAGTCTCCTCAATGTCATCCTCTGAACTG GTCCACTTGTAGCCCTGCTTTGACATCCAGGCAGCnTaCAGGTGTGATG ACGTCCTCAGACCCGCCAGACACATCCGATCCAAAGAGCCTCATCACC TTGAACGTCGTGTTCGTCATCATCGTCATGGTAGTTGTGCCTGGG TTATGTTGTAGCCGACCGTGCTCAGGCTCTCACGGTGGATGGTT CTTGCTTTCATGCATTGTGCTTCACTTTTGTTCTAATCTAGGATGG GTTGGATCTTCTTATAGTTTTGGATCGATGATTCAAGAGAGTCGA TGTGCAACTCTCCTACATATGGTTAGGAAAAAACTATGAACACTA GGGGCAGGGTGTCAAACTAGTGGGGAAGCTTTTCTAGTCAACTG CCCCCACGTCGCTGCCTAGGCGACTCAGGCGGCCACCCATCCT CGTCGCCGGAGGGTGGTCCTCACTTCCCTCCCCTCATCGTCATT GTCGATTGACGCCACCGGGGATCTGCCCTAGAGGACGATGGTG GTGGCGAGCCATCTTCCTCTCGTCCTACATGCTCCGACATCCTTT CTGATGAGGCCTCCCTATCGGTTTCGCCTACATGAAGCTA TTCTGTCACGGCGGAGACTCTCCTGTCAGTCAC Sequences of amplified product located at 2** bp4 Sequences of amplified product located at /** bp4 Fig.. Application of wheat-specific STS primer to commercial food products Abbreviations : +, Bread ;,, Noodle ; -, Chinese noodle ;., Soumen ; /, Strong flour ; 0, Cake flour, M ; Molecular weight marker. UNI,f K2** 2** bp K/** /** bp./* bp *.*+ PCR - K2** K/** Fig.. K2** 2** bp K/** /** bp./* bp K2** K/** K/** K/** PCR DNA PCR PCR +2S rdna PCR +/ K/** +2S rdna DNA +,* +-* PCR DNA PCR

5 86 /.,,**1, 26 PCR UNI,f VIEr 2** bp /** bp STS K/** DNA K/** +, -. / Quinteiro, J., Sotelo, C.G., Rehbein, H., Pryde, S.E., Medina, I., Perez-Martin, R.I., Rey-Mendez, M. and Mackie, I.M., Use of mtdna direct polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism methodologies in species identification of canned tuna. J. Agric. Food. Chem.,.0, +00,+003 (+332). Martinez, I. and Danielsdottir, A. K., Identification of marine mammal species in food products., J. Sci. Food Agric, 2*, /,1/-- (,***). Mihalov, J.J., Marderosian, A.D. and Pierce, J.C., DNA identification of commercial ginseng sample. J. Agric. Food. Chem.,.2, /, (,***). Miguel, A.P. and Begona, P.-V., Identi cation of commercial canned tuna species by restriction site analysis of mitochondrial DNA products obtained by nested primer PCR. Food Chem., 20, +.-+/* (,**.) ,**. 0 Saez, R., Sanz, Y. and Toldra, F., PCR-based fingerprinting techniques for rapid detection of animal species in meat products. Meat Science, 00, 0/300/ (,**.). 1 Maria, L.-A., Laura, L., Amando, G.-P., M. Isabel P. and Antonio, P, Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction. Anal. Biochem., --3, 1-2, (,**/). 2 RAPD,3.1/*,**- 3 Uchino, M., Noguchi, T. and Takano, K., Thermostability of DNA from wheat in heated food products. Food Pres. Sci., -*, +3/+32 (,**.). +* Saito, H. and Miura, K., Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochem. Biophy. Acta, 1,, 0+30,3 (+30-). ++ Watson, J.C. and Thompson, W.F., Purification and restriction endonuclease analysis of plant nuclear DNA. In Methods for Plant Molecular Biology. eds. Weissbach, A. and Weissback, H., Academic Press, Inc, pp. /110 (+322). +, Okada, G., Takematsu, A. and Takamura, Y., Phylogenetic relationships of the hyphomycete genera Chaetopsia and Kinochaeta based on +2S rdna sequences. Mycoscience, -2,.*3.,* (+331). +- Tateno, Y., Saitou, N., Okubo, K., Sugawara, H. and Gojobori, T., DDBJ in collaboration with masssequencing teams on annotation. Nucleic Acids. Res., --, D,/-D,2 (,**/). +. Thompson, J.D., Higgins, D.G. and Gibson, T. J., CLUSTAL W : improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res,,,, * (+33.). +/ DNA PCR Polymerized Chain Reaction,3 --/--2,**- +2 0,0 +2 +*,0

474 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /-, No.3,.1..2* (,**0) 24 Measurement of Deterioration of Frying Oil Using Electrical Properties Yoshio

474 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. / No.3.1..2 (0) 24 Measurement of Deterioration of Frying Oil Using Electrical Properties Yoshio Hagura Yoshihiro Sasaki and Kanichi Suzuki Graduate School