- Email: [email protected]
Microarray detection for eighteen events of genetically modified organisms
S250
Abstracts / Journal of Biotechnology 136S (2008) S247–S251
low to 10 copies and 20 copies canola genomic DNA in quantitative PCR assay, respectively. Furthermore, the ideal quantified results were obtained in the practical canola samples detection. All the results indicated that the developed qualitative and quantitative PCR methods based on the revealed 3’ integration flanking sequence are suitable for GM canola Oxy-235 identification and quantification. Reference Holst-Jensen, A., Rønning, S.B., Løvseth, A., Berdal, K.G., 2003. PCR technology for screening and quantification of genetically modified organisms (GMOs). Anal. Bioanal. Chem. 375, 985–993.
doi:10.1016/j.jbiotec.2008.07.532 IV4-P-006 Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of roundup ready soybean Haibo Zhang, Litao Yang, Jinchao Guo, Dabing Zhang ∗ GMO Detection Laboratory, School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China E-mail address: [email protected] (D. Zhang). To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs (Yang et al., 2007). However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis (Taverniers et al., 2001; Kuribara et al., 2002). In this study, we developed one unique plasmid molecule based one pMD18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), i.e. CaMV35s, NOS and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting to CaMV35s, NOS and RRS event employing this reference molecule as calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 copies and 50 copies, respectively. For the quantitative analysis of practical RRS samples, results of the accuracy and precision were similar to those of singleplex PCR assays, for instance, the quantitative results at 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6% respectively, and statistic analysis (t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction, and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs. References Kuribara, H., Shindo, Y., Matsuoka, T., Takubo, K., Futo, S., Aoki, N., Hirao, T., Akiyama, H., Goda, Y., Toyoda, M., Hino, A., 2002. Novel reference molecules for quantitation of genetically modified maize and soybean. J. AOAC Int. 85 (5), 1077–1089. Taverniers, I., Windels, P., van Bockstaele, E., De Loose, M., 2001. Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products. Eur. Food Res. Technol. 213, 417–424.
Yang, L.T., Guo, J.C., Pan, A.H., Zhang, H.B., Zhang, K.W., Wang, Z.M., Zhang, D.B., 2007. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule. J. Agric. Food Chem. 55, 15–24.
doi:10.1016/j.jbiotec.2008.07.533 IV4-P-011 Microarray detection for eighteen events of genetically modified organisms Jae-Hwan Kim ∗ , Su-Yeon Kim, Hae-Yeong Kim Graduate School of Biotechnology, Kyung Hee University, Yongin 446701, Republic of Korea E-mail address: [email protected] (J.-H. Kim). A DNA microarray chip (Xu et al., 2005; Leimanis et al., 2006; Xu et al., 2006; Xu et al., 2007) was developed for the detection of 18 genetically modified organisms (GMOs), which are two GM soybeans (GTS-40-3-2 and A2704-12), twelve events of GM maize (Bt176, Bt11, MON810, MON863, NK603, GA21, T25, TC1507, Bt10, DAS59122-7, TC6275, and MIR604), three events of GM canola (GT73, MS8xRF3, and T45), and one GM cotton (LLcotton25). The microarray include a total of 26 oligonucleotide probes for endogenous reference targets, event-specific targets, screening targets (35S promoter and nos terminator), and internal targets (18S rRNA). The genes corresponding to lectin, starch synthase IIb (SSIIb), fatty acyl-ACP thioesterase (FatA), and acyl carrier protein (Acp1), were chosen as endogenous reference genes of soybean, maize, canola, and cotton, respectively. To simultaneously detect multiple target sequences in GMOs, multiplex PCR was coupled with a microarray and the designed primer pairs successfully amplified the target sequences. The limits of detection are 0.5% for GTS 40-3-2 soybean and 1% for MON810 maize. This result showed that the microarray system would play an important role in detection of GMOs in a variety of food ingredients including soybean, maize, canola, and cotton. References Leimanis, S., Hernández, M., Fernández, S., Boyer, F., Burns, M., Bruderer, S., Glouden, T., Harris, N., Kaeppeli, O., Philipp, P., Pla, M., Puigdomènech, P., Vaitilingom, M., Bertheau, Y., Remacle, J., 2006. A microarray-based detection system for genetically modified (GM) food ingredients. Plant Mol. Biol. 61, 123–139. Xu, X., Li, Y., Zhao, H., Wen, S.Y., Wang, S.Q., Huang, J., Huang, K.L., Luo, Y.B., 2005. Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray. J. Agric. Food Chem. 53, 3789–3794. Xu, J., Miao, H., Wu, H., Huang, W., Tang, R., Qiu, M., Wen, S., Li, Y., 2006. Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray. Biosens. Bioelectron. 22, 71–77. Xu, J., Zhu, S., Miao, H., Huang, W., Qiu, M., Huang, Y., Fu, X., Li, Y., 2007. Eventspecific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray. J. Agric. Food Chem. 55, 5575–5579.
doi:10.1016/j.jbiotec.2008.07.534
Abstracts / Journal of Biotechnology 136S (2008) S247–S251
low to 10 copies and 20 copies canola genomic DNA in quantitative PCR assay, respectively. Furthermore, the ideal quantified results were obtained in the practical canola samples detection. All the results indicated that the developed qualitative and quantitative PCR methods based on the revealed 3’ integration flanking sequence are suitable for GM canola Oxy-235 identification and quantification. Reference Holst-Jensen, A., Rønning, S.B., Løvseth, A., Berdal, K.G., 2003. PCR technology for screening and quantification of genetically modified organisms (GMOs). Anal. Bioanal. Chem. 375, 985–993.
doi:10.1016/j.jbiotec.2008.07.532 IV4-P-006 Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of roundup ready soybean Haibo Zhang, Litao Yang, Jinchao Guo, Dabing Zhang ∗ GMO Detection Laboratory, School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China E-mail address: [email protected] (D. Zhang). To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs (Yang et al., 2007). However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis (Taverniers et al., 2001; Kuribara et al., 2002). In this study, we developed one unique plasmid molecule based one pMD18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), i.e. CaMV35s, NOS and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting to CaMV35s, NOS and RRS event employing this reference molecule as calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 copies and 50 copies, respectively. For the quantitative analysis of practical RRS samples, results of the accuracy and precision were similar to those of singleplex PCR assays, for instance, the quantitative results at 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6% respectively, and statistic analysis (t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction, and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs. References Kuribara, H., Shindo, Y., Matsuoka, T., Takubo, K., Futo, S., Aoki, N., Hirao, T., Akiyama, H., Goda, Y., Toyoda, M., Hino, A., 2002. Novel reference molecules for quantitation of genetically modified maize and soybean. J. AOAC Int. 85 (5), 1077–1089. Taverniers, I., Windels, P., van Bockstaele, E., De Loose, M., 2001. Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products. Eur. Food Res. Technol. 213, 417–424.
Yang, L.T., Guo, J.C., Pan, A.H., Zhang, H.B., Zhang, K.W., Wang, Z.M., Zhang, D.B., 2007. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule. J. Agric. Food Chem. 55, 15–24.
doi:10.1016/j.jbiotec.2008.07.533 IV4-P-011 Microarray detection for eighteen events of genetically modified organisms Jae-Hwan Kim ∗ , Su-Yeon Kim, Hae-Yeong Kim Graduate School of Biotechnology, Kyung Hee University, Yongin 446701, Republic of Korea E-mail address: [email protected] (J.-H. Kim). A DNA microarray chip (Xu et al., 2005; Leimanis et al., 2006; Xu et al., 2006; Xu et al., 2007) was developed for the detection of 18 genetically modified organisms (GMOs), which are two GM soybeans (GTS-40-3-2 and A2704-12), twelve events of GM maize (Bt176, Bt11, MON810, MON863, NK603, GA21, T25, TC1507, Bt10, DAS59122-7, TC6275, and MIR604), three events of GM canola (GT73, MS8xRF3, and T45), and one GM cotton (LLcotton25). The microarray include a total of 26 oligonucleotide probes for endogenous reference targets, event-specific targets, screening targets (35S promoter and nos terminator), and internal targets (18S rRNA). The genes corresponding to lectin, starch synthase IIb (SSIIb), fatty acyl-ACP thioesterase (FatA), and acyl carrier protein (Acp1), were chosen as endogenous reference genes of soybean, maize, canola, and cotton, respectively. To simultaneously detect multiple target sequences in GMOs, multiplex PCR was coupled with a microarray and the designed primer pairs successfully amplified the target sequences. The limits of detection are 0.5% for GTS 40-3-2 soybean and 1% for MON810 maize. This result showed that the microarray system would play an important role in detection of GMOs in a variety of food ingredients including soybean, maize, canola, and cotton. References Leimanis, S., Hernández, M., Fernández, S., Boyer, F., Burns, M., Bruderer, S., Glouden, T., Harris, N., Kaeppeli, O., Philipp, P., Pla, M., Puigdomènech, P., Vaitilingom, M., Bertheau, Y., Remacle, J., 2006. A microarray-based detection system for genetically modified (GM) food ingredients. Plant Mol. Biol. 61, 123–139. Xu, X., Li, Y., Zhao, H., Wen, S.Y., Wang, S.Q., Huang, J., Huang, K.L., Luo, Y.B., 2005. Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray. J. Agric. Food Chem. 53, 3789–3794. Xu, J., Miao, H., Wu, H., Huang, W., Tang, R., Qiu, M., Wen, S., Li, Y., 2006. Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray. Biosens. Bioelectron. 22, 71–77. Xu, J., Zhu, S., Miao, H., Huang, W., Qiu, M., Huang, Y., Fu, X., Li, Y., 2007. Eventspecific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray. J. Agric. Food Chem. 55, 5575–5579.
doi:10.1016/j.jbiotec.2008.07.534