Quantitative Analysis of Genetically Modified Soybean in Processed Foods Using Real-time PCR

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 36, No. 5, pp. 723~727 (2004). ©The Korean Society of Food Science and Technology. *%Î²ê>wj Ï F*¶æ; &®~ ;ïªC ÿ«1,2ÁB>2Á;*¢1Áî^+2ÁBê]1ÁB2,* ã³Ö>®î&Òö þ²1, ã\v ®«"2. Quantitative Analysis of Genetically Modified Soybean in Processed Foods Using Real-time PCR Dong-Myung Min1,2, Myo-Young Kim2, Soon-Il Jung1, Mun-Seok Heo2, Jin-Kuk Kim1, and Hae-Yeong Kim2,* 1. National Agricultural Products Quality Management Service, Ministry of Agriculture 2 Department of Food Science and Biotechnology, Kyung Hee University. Qualitative and quantitative PCR methods were performed to examine detection and quantitation of epsps inserted into genetically modified soybean (GMS) in processed foods, soy milk, tofu, and biji (soybean curd residue). Using PCR amplification to produce two (121 and 330 bp) epsps in GMS, detection limits of GMS in soy milk, tofu, and biji containing 0.01% GMS were measured. For quantitative detection, test samples containing 1, 3, and 5% GMS were measured by real-time PCR method. Results show real-time PCR method is applicable to detect GMS quantitatively in processed foods. Key wards: EPSPS, genetically modified soybean, PCR, TaqMan7700, quantitative PCR. B. . Û" "' ¦;O»~ BB~ jºW º>î . GMO~ "º ¦ÒO»bº 7Î²ê>w(polymerase chain reaction, PCR) B &ªC»b ºÂ> ®b, ÚöBê F*¶æ; ~ ¦;»(6-8)öBº PCRj Ï ªCO» ;ã>Ú GMO~ ¦;O»b Ï> ® . ¾, GMOj Ï &®~ ãÖ öò¢ Ï~ ; ïªCj ~ ®Ú ¢¦~ ÖJ& ®b ¢ &j~ Ö Ò¾¢öBê GMOf &®ö & ;W, ;ï¦Ò»~ BB 5 &z & B® Úæ ® . öBº GMj öò &®(v¦, vF, jæ) ö &~ B";öB~ 5 " ö ~ &ö ~ DNA æW ;W 5 ;ï¦;ö âî¾ 'Ëj ~ºæ¢ {~V *~ PCR ÃÖb V& 2«~~ primer¢ Ï~ ;W¦;ê G; þj ~&b, &®ö & ;ï¦; æzç¢ G;~ ¦;&ËWj {~ .. V~ vÇö ~ ®« G«~ ê¢ ~V * & nb «VFj Ï~ FÏ F*¶¢ ê«Î F *¶æ; ³·b BB 5 çë' ÒVf FÛ /³® {& > ® . F*¶æ;·b(genetically modified organism, GMO)f ÒV~ ÎNWb * ^ê'b ÒV' 2003j 6,770ò ha& ÒV> ®b º *j&j 15% ;ê Ã& ©b î v ¶Ò > ç~ Ã&Nj ®, çëzB GMOê 2003jö V& 18B ®« 78B~ ·bö ~ ® (1). Þbº F*¶æ;³Öb Ú, ~ã, Ò² ' FWö & ¢¦ Ú'b pª ì BV>º ç öB ÖÒ¾¢¢ j EU, ¢ ^ê 'öB F*¶ æ;³Öb B¢ ê«~ Ú'~ ® . F*¶æ;³Öbf ªêFÛj ~z¢ê 2«, ÒV, >{, FÛ";öB j~ê'b GMOf non-GMO~ b« Úæº 6j 6n BöBº j~ê' b«~¢ J ;~ Ú'~ ® . EU~ ãÖº 0.9%, ¢, &òf 5.0%, ÖÒ¾¢º 3.0% J;~ Ú'~ ® (2-5). B~ ³ ;O" ÎN' &Ò¢ *B Úê' ªêF. Òò 5 O» Òò Non-GMf ³ÖbFÛÒöB >«B Ö j ;W ªC~ GMO& F>æ pf ©j { ê, non-GM b Ï~&b, GM f SDI ²Ò¦V Glyphosate &W (Roundup Ready Soybean)j /Aj ªêV(RotorSpeed Mill P14, Fritsch GembH, Germany)~ 500 µm çÚ¢ Ï~ «¶~ V& 500 µm ~ ¢~² ªê~ Ò Ï~& . ªêB ªöj GM" non-GMj '' Z²jN. *Corresponding author: Hae-Yeong Kim, Department of Food Science and Biotechnology, Kyung Hee University, Suwon 449-701 Korea Tel: 82-31-201-2660 Fax: 82-31-201-2157 E-mail: hykim@khu.ac.kr 723.

(2) ®"²æ B 36 ² B 5 ^ (2004). 724. b ê ªêbb Ïª® b~ ¦;ê 5 ; ï¦; G;Ï ò ÒÏ~& . &®B þ~ ãÖ ;{ b«jNj ò V *~ 500 µm ªêB ªöj ò Ú ÒÏ~& . ªö 100 gö Ã~> 1.7 Lj I 20ª* &~& . ® ê ê jæ ¢ ò j~& . ®¢ Û" vF¢ j~ ò ÒÏ~& . v¦º vFö "zîJ¾(MgCl2)j Î&~ & f ê 5ª* O~~ wÎ òj Ò'æö I Â÷ b 2 v¦¢ ò Ú ò ÒÏ~& . ''~ òöB vB~ þ2j j~ ò ÒÏ~& .. Fig. 1. Schematic diagram of (A) primers for detection of GM soybean and (B) standard plasmid pMulSL2 as reference molecule.. ò % DNA ºÂ" *ê 5 ³ê /; DNA ºÂf QiagenÒ~ DNeasy Plant Mini Kitj ÒÏ~. r" ?f O»j Ï~& . ò 1 g¢ AP1 buffer 10 mL f RNase A(100 mg/mL) 20 µLj Î& ê î~² DÚ 65 öB 1*ÿn N¾Ò~B 15ªî 10.* vortexê N öB 10ª* 3,000Üg öªÒ ê ç[(7 mL)j ~ î tubeö TV AP2 buffer 2.5 mL¢ IÚ 10.* vortex. r âr ³öB 15ª* ïN ¾Ò~& . NöB 35ª* ö ªÒ ê ç[ 8 mL¢ QIA shredder spin columnö I 5 ª* öªÒ~ Û" Ï 6.8 mL¢ îÚ Â2ö Tâ. . öêR FB AP3 buffer 10.2 mL¢ Î&~ 10.* vortex r DNeasy spin columnö I NöB 15ª* ö ªÒ ê ¢"j Û" Ïf ª^ . öêR FB AW buffer 12 mL¢ ¢"ö I NöB 15ª* öªÒ ê ¢"j îÚ 50 mL Â2ö TV Ò 65oC &N > 1 mLj Î&~ NöB 5ª* ¾Ò~& . NöB 10 ª* öªÒ~ ÏÂB DNAÏj 2 mL Â2ö TZ ê, ÿï~ isopropanolj I b ê NöB 5ª* ¾Ò~&. . 4oCöB 15ª* 13,000Üg öªÒ~ ç[f ª Ò 70% öêR 0.5 mL DNA pelletj af r, 4oC öB 3ª* 13,000Üg öªÒ~& . öªÒ ê~ ç[ f ªÒ, DNA pelletf Î r NÃ~> 100 µLö ö DNA ÒÏ~& . ºÂ DNA Bê 5 ³êº j& Ê ºj Ï *V'ÿ»" ª77êê»j ÒÏ~ 230, 260 5 280 nmöB~ 7ê¢ G;~& .. ;W PCR ªC PCR ;WªCö ÒÏ primerf ;º Fig. 1Af Table 1ö º£~&b, PCR >wÏ(Taq DNA Polymerase 1 unit, MgCl2 2.5 mM, dNTPs ' 200 µM, KCl 50 mM, Tris-HCl(pH 8.0) 10 mM)f AmpliTaq Gold DNA Polymerase, GeneAmp 10X PCR Buffer, GeneAmp dNTP Blend(Applied Biosystems, USA)¢ ÒÏ~& . PCRf DNA 100 ngj ";b primer¢ 50 ngO Î&~ *Ú 20 µL~ >wb 94oCöB 10ª* æW >w ê, 94oCöB 30., 60oCöB 30., 72oC öB 30.~ ¢N~ >wj 40² ê, 72oCöB 10ª* ~ 7>wj º&~ 4oC ï'~ «ò~& (4). PCR >wöBº non-GM &òöB ºÂ DNA¢ ÚÒ F*¶ 5 ê«F*¶ö & & ÷¯~ ªC~& . PCR ê~ Öbf 2% j&Ê ºj Mupid *V'ÿV(Advance, Japan)ö ËO~ 100(voltage)öB 20ª* *V'ÿ~ CCD z¢ R'~ {~& . PCR VVº GeneAmp PCR system 9700(Applied Biosystems, USA)¢ ÒÏ~& .. *8'ÿ Ï» 1% j&Ê ºj Mupid *V'ÿV(Advance, Japan)ö J. ;ï PCR ªC þöB ÒÏB Real time-PCR &bîf GM(RRS). ~ ê ò¦V ºÂ DNA ö 5 µLö *V'ÿÏ loading buffer(0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll) 1 µLj b~ loading ê 100 volt *{ (5 V/cm)b 20ª ÿn *V'ÿ~ UV transilluminator *ö ºj RJ CCD z¢ b R'~ zçV ~ & (9). *V'ÿ Ö" DNA~ ª¦, RNA 5 ~ ~ bÒ ¦ ºÂ DNAö & Bê¢ {~& .. Table 1. List of PCR primers and probes in this study Primer (Gene). Sequence (5' 3'). Specifticity. Lec01-5'&3' (Soy endogeneous). GCC CTC TAC TCC ACC CCC A GCC CAT CTG CAA GCC TTT TT (Fam)-AGC TTC GCC GCT TCC TTC AAC TTC AC-(Tamra). Sense Antisense Probe. 35S2A&CTP5A (Target). ATG ACG CAC AAT CCC ACT ATC GGC TGT AGC CAC TGA TGC TGA. Sense Antisense. C-EP01-5'&3' (Target). CCT TTA GGA TTT CAG CAT CAG TGG GAC TTG TCG CCG GGA ATG (Fam-CGC AAC CGC CCG CAA ATC C-(Tamra). Sense Antisense Probe. Length (bp). 118. 330. 121.

(3) &®öB~ F*¶æ; ~ ;ï ªC. 725. Fig. 3. Genomic-DNA patterns isolated from soy food. Lanes 1-2: soybean, lanes 3-4: soymilk, lanes 5-6: tofu, lanes 7-8: biji, M: 1kb Ladder (Sigma-aldrich, USA). Fig. 2. Amplification plots and standard curves for real-time PCR. (A) Amplification curves (6 doses, 0, 20, 125, 1,500, 20,000, and 250,000 copies of pMulSL2) were generated for detection of GM soybean and each curve corresponds to three replicates. (B) Parameters of the regression line through data points are indicated within the plot. The slope of regression line is -3.503.. 1êÛj ¦Â > ®º *¢^f ÚÒF*¶ *¢^¢ Ï~ ÃB ' PCR Öb(amplicon)j ~¾~ plasmidö ã «~ Ö amplicon plasmid(Fig. 1B)¢ ÒÏ~& . , amplicon plasmidº &ò~ B ÚJÞ r, BB plasmid¢ Ã~ £² &ò ~Ö~ Ï~º O»b BB>î (10,11). Amplicon plasmidº Fig. 2öB º:f ? salmon testis DNA(Sigma, USA, 5 ng/µL) 2.5 µL 0, 20, 125, 1,500, 20,000, 250,000 copies C~ &bî ÒÏ~& . Real time-PCRf ªCÏ ò 16 3 well > ªC~, well >wÏf 25 µL ~& . ÚÒ F*¶ 5 ê«F*¶ ªCÏj '' &j~& . >wÏj * primer/probe mix (NipponGene, Japan)º ''~ primer 1.25 µM, TaqMan® probe 0.5 µM¢ b~ ò î . -² ò Úê primer/probe mixf TaqMan® universal PCR master mix (Applied Biosystems, USA)º 1 : 1.25~ jN >wÏj B~& . >wÏ Wf TaqMan universal PCR master mix 12.5 µL, primer probe mix 10 µL, Template DNA(20 ng/µL) 2.5 µL b~&, centrifuge spin down ~ PCRj >¯ ~& . PCR >wf 95oC 30., 59oC 1ªb ~ 40 cyclej >¯~& . PCR &º DNA¢ Iæ pf rW& salmon sperm DNA(5 ng/µL)¢ IÚ ÒÏ~& . ;ïªCÖ"~ ªC. þ Â Vº ê«F*¶f ÚÒF*¶ ¾*Ú sequence detection systems 1.9.1 programj Ï~ ªC~&. . ÃN(A) 5 | ∆A |¢ êÖ~ j òÊº threshold linej Ö; ê ªCò~ ÚÒF*¶ 5 ê«F* ¶ B>(copy)¢ {~& . A8f 10(-1/slop), | ∆A | = (Am + 1 − Am)/AmÜ100b, r | ∆A |& 2* ç ³~ 1% ~& >º *(²m, &m)~ 7*æ6(mt)j threshold lineb Ö;~& . b«Nf r~ j ÒÏ~ êÖ~&b, B.B&W (RRS)~ ;ê> 0.95¢ ¢'b 'Ï~& . b«N(%) = ªCò~ GMêÛ ß ê«F*¶ > 1 Ü Ü100 ªCò~ ÚÒF*¶~ > ;ê>. Ö" 5 8 DNA *ê/; öò , vF, v¦, jæöB DNA¢ ºÂ Ö" & ö V¢ · DNA patternj & . Fig. 3öB º : f ? öò f DNA& ² ¶ç>æ pf ç ºÂ> îb¾ vFf v¦~ ãÖº & ";öB ö ~~ ª ¶ï DNAº ç>& ª >îrj { > ®î .. ò, jæ~ ãÖöº ºÂB genomic DNA& öò º ô ª>îæò, &";öB ª>æ pf ^öB º ÂB ª¶ï~ DNA& ¢¦ ºÂB ©b {>î . PCR ;W¦; &®~ ãÖ &";öB , " ~ ¾Òö ~~ Fig. 3öB º :f ? template ÒÏF DNA& ô ª> ö V¢ PCR ÎNö 'Ëj ©b .GF > ®. . ß®, ®~£®n*Ó F*¶Ò®¦ÒæöBº PCR Öb 200 bp çb *¢^¢ Jê~º ãÖ & ® ªCö 'æ pf ©b > ®Ú, þöBº vF, v¦~ ãÖöê PCR Öb~ Vö V 'Ë ¾æ ¾º æ¢ {~& . Table 1~ v«~~ *¢^¢ Ï~ PCR Öb~ 121 bpf 330 bp V& W¦¢ {~ ¦;ê~¢ '' G;~& . Fig. 4f 5öB º :f ? vF, v¦, jæ Î òöB 121 bpf 330 bp Öb 0.01%ræ ¦Â &Ë~& . Ö" " r vF, v ¦º ~ &®Vº ~¾ &"; B~ ö ò¢ b~º &® jîÚB ¦Â 6ê& öò" N& ìº ©b ÒJB . PCR ;ïªC 1, 3 5 5%~ GM '' B jæ, vF, v¦öB ~ ÚÒF*¶ lectin" ã«F*¶ epsps F*¶~ ¦ÂN " jv~ &®öB~ ;ï&ËWj þ~& . Table 2öBf ? öò 5 &®~ ãÖ vF¢ B v¦ f jæöB 1% GM òº 0.88-0.99%, 3% GM ò º 2.7-3.2%, 5% GM òº 4.21-5.29% ª~& . ö ò , v¦ 5 jæöB~ &JNº −16-7%, &ÞNº 6.25-13.24% n;' ©b ¾æÒ . öò öB GM j Nf '' 0.99Û0.13, 3.20Û0.30 5 5.29Û0.36% ¾æ¾ b «jN ¸f ©b 6;>îb¾ ÞN¢ J~ Ï' 8~ º*Úö ³~º ©b ¾æÒ . &®~ ãÖ vFº 0.74Û0.11, 1.89Û0.18 5 3.12Û0.23% ¾æ¾ *Ú'b b.

(4) 726. ®"²æ B 36 ² B 5 ^ (2004) Table 2. Results of quantitative test with different GMO ratio Accuracy Bias True value Average (%) GMO True value (%) (%). Fig. 4. Sensitivity analysis of PCR for detection of various amounts of RRS with C-EP01-5 and 3. (I) soybean (II) soymilk (III) tofu (IV) biji, Lane 1: Non-GM soybean, lanes 2-3: 0.01% RRS, lanes 4-5: 0.05% RRS, lanes 6-7: 0.1% RRS, lanes 8-9: 1.0%, lanes 10-11: 3.0% and lanes 12-13: 5.0% RRS, M: PCR Marker (Sigma-aldrich, USA).. Fig. 5. Sensitivity analysis of PCR for detection of various amounts of RRS 35S2A and CTP5A. (I) soybean (II) soymilk (III) tofu (IV) biji. Lane 1: Non-GM soybean, lanes 2-3: 0.01% RRS, lanes 4-5: 0.05% RRS, lanes 6-7: 0.1% RRS, lanes 8-9: 1.0%, lanes 10-11: 3.0% and lanes 12-13: 5.0% RRS, M: PCR marker.. «jN 20-30% ;ê Ô² ¾æÒb, v¦öBº 0.89Û 0.09, 2.86Û0.34 5 3.69Û0.56% 10% ;ê Ô² ¾æÒ j æ~ ãÖº '' 0.88Û0.12, 2.70Û0.17 5 4.21Û0.26% ¾ æ¾ &®f *Ú'b öò Ô² ¾æ¾º ãËj ¾æÚî . v¦, jæº ;ï¦; &Ë ©b {>îb¾, vF ~ ãÖ GM ~ ï F*¶~ B bjN º Ô² ¾æ¾ ;ï ÚJÚ ©b {>î . ¾ b«jN Ã &ö V ;ï¦;F~ çFW (r2)f 0.9997 ç&W ¸² ¾æ¾ ;ï ¦;~~ ;b ;ï &Ë ©b 6> î . %FÛ L¦öB~ ÎîVç Real time PCRj Ï &® &Ú v¦ö &. Precision SD (%). RSD (%). Soy bean. 1.0 3.0 5.0. 0.99 3.20 5.29. -1.00 -6.67 -5.80. 0.13 0.30 0.36. 13.13 09.31 06.81. Soy milk. 1.0 3.0 5.0. 0.74 1.89 3.12. -26.00 -37.00 -37.60. 0.11 0.18 0.23. 14.24 09.58 07.41. Tofu. 1.0 3.0 5.0. 0.89 2.76 4.29. -11.00 0-8.88 -14.20. 0.09 0.33 0.29. 09.59 12.06 06.69. Biji. 1.0 3.0 5.0. 0.88 2.70 4.21. -12.00 -10.00 -15.80. 0.12 0.17 0.26. 13.32 06.30 06.25. ;ïO»ö & þÖ"& F~W ®² ¾N þÖ"¢ : ûb 2003jêö >÷B v¦ò j Ï~ FÛ * " GM ~ b«Nj ;W 5 ;ï PCRj Û~ >¯~& . v¦ò f &®ö & öÖæ &Ò¢ * O»b öò ~ Öæ& ÚÖb B B®j &çb 2003j 5ú¦V 6úö ÷7'b ò¢ j~& . * 'b FÛ>º 946~ ò¢ j~, ;W PCR O»b GM ¦¢ Fê~ 226 ·Wb ¦Â>îb, ·W b 6êB 226ö & real time PCRj Ï~ GM b«Nj G; Ö" Î ò& 3% òb 3%¢ ."~ º òº *& ìº ©b {>î . ~ ãÖ B *öº GM ~ b«N 3%¢ ." ~&æò, B& ¯B 2003jöº ®&Ï ~ ãÖ 3% ~ FÛB º (8)f &®~ ãÖê B& ¯B 2001j 7ú *öº ~ Î &®öB GMO& ¦Â>îb¾ B êöº &¦ª ¦Â >æ p f ©b (12)f FÒ Ö"¢ ®rj {~& .. º. £. &® vF, v¦, jæöB F*¶æ; ö ã« B epsps F*¶~ ¦Â" ;ïj * ;W" ;ï PCR O» j >¯~& . vF, v¦, jæöB F*¶æ; ~ ¦Âf ã«B epsps F*¶~ Ã V& 121 bpf 330 bp& W F > ®º v «~~ primer j Ï~ 0.01%ræ {~ & . ;ïO»f 1, 3, 5%~ F*¶æ; B ò. j * PCRj ÒÏ~ F~W ®º Ö"¢ áî . Ö" f * PCR O»j ÒÏ~ &® ÚöB ; ï'b F*¶æ; j ;ï~º 'ÏF > ®rj & .. 6Ò~ "Bº ã³Öb®î&Òö GMO¦;» BB "Bf ³2êÓ :Ö 21Òë "B~ j æ öj Aj >¯>îÛî . ö 6Òãî ..

(5) &®öB~ F*¶æ; ~ ;ï ªC. ^. ò. 1. James C. Preview: Global Status of Commercialized Transgenic Crops: ISAAA Briefs No. 30. ISAAA, Ithaca, NY, USA (2003) 2. Auer CA. Tracking genes from seed to supermarket: techniques and trends. Trends Plant Sci. 8: 591-597 (2003) 3. Ahmed FE. Detection of genetically modified organisms in foods. Trends Biotechnol. 5: 215-223 (2002) 4. Korea Ministry of Agriculture and Forestry. Regulation Concerning the Compulsory Labeling of Genetically Modified Agricultural Organisms. Korea Ministry of Agriculture and Forestry, Seoul, Korea (2000) 5. Korea Food and Drug Adminstration. Regulation Concerning the Compulsory Labeling of Foodstuffs and Food Ingredients Produced from Genetically Modified Organisms. Korea Food and Drug Adminstration, Seoul, Korea (2001) 6. Kim HJ, Park SH, Kim HY. Study for detection of glyphosate tolerant soybean using PCR. Korean J. Food Sci. Technol. 33: 521-524 (2001) 7. Kim YM, Sohn SH, Jeong SI, Yoon MS, Kim TS, Park YH. Detection methods for genetically modified soybeans. J. Korean Soc. Agric. Chem. Biotechnol. 45: 185-189 (2002). 727. 8. Jung S. Studies on the detection of genetically modified organism using polymerase chain reaction. MS thesis. Kongju University, Kongju, Korea (2004) 9. Kim JH. Studies on the polymerase chain reaction for the rapid detection of genetically modified foods. MS thesis. Kyunghee University, Suwon, Korea (2003) 10. Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N, Hirao T, Akiyama H, Goda Y, Toyoda M, Hino A. Novel reference molecules for quantitation of genetically modified maize and soybean. J. AOAC Int. 85: 1077-1089 (2002) 11. Shindo Y, Kuribara H, Matsuoka T, Futo S, Saqada C, Shono J, Akiyama H, Goda Y, Toyoda M, Hino A. Validation of real-time PCR analysis for line-specific quantitation of genetically modified maize and soybean using new reference molecules. J. AOAC Int. 85: 1119-1126 (2002) 12. Kim MY, Kim JH, Kim HJ, Park SH, Woo GJ. Kim HY. Monitering of genetically modified soybean and processed foods in Korean market using PCR. J. Korean Soc. Agric. Chem. Biotechnol. 46: 344-347 (2003) (2004j 7ú 27¢ %>; 2004j 8ú 27¢ j).

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