Development of a multiplex and semi-quantitative PCR assay with restriction enzyme reaction for detection of genetically modified Bt maize in food

• Main Authors: Chi-Lung Chiu, 邱啟隆
• Other Authors: Tony Ji Fang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/72590159022602057362

碩士 === 國立中興大學 === 食品科學系 === 92 === Numerous analytical methods, both qualitative and quantitative, have been developed to determine reliably the presence and/or the amount of transgenes in agricultural commodities, in raw agricultural materials and in processed and refined ingredients. In this study, a semi-quantitative PCR methology be developed. First, to design and collect referenced primers, and then to compare the efficiency of each pair primers. Two pairs primers, ivr1-1/ivr1-2 and 35SP-01/35SP-02, both that were used in multiplex PCR. 5% MON810 standard was detected and parameters constracted by multiplex with ivr1-1/ivr1-2 and 35SP-01/35SP-02. Following the principle of quantitative competitive PCR (QC-PCR), and as regards maize endogene as competitor. According to the screening, which could get a parameter of constant ratio. However, the coefficient of variation would become more higher than 25% (European inter- laboratories test results). So, the template DNA mass range must be fixed betwen 10 ng to 100 ng. Transgene contents were calculated by multiplex PCR methology with ivr1-1/ivr1-2 and 35SP-01/35SP-02 primers in first detective step. Secondly, the cry1A(b) gene content be detected by simplex PCR with cryIA4-5’/cryIA4-3’ primers. And RsaI restriction endonuclease can distinguish with three Bt maize (MON810, Bt11 and Event176) at right after. Analysis of maize endogene by simplex PCR with ivr1-1/ivr1-2 primers in precessed food. Injury data show that maize DNA would breakdown and make low sceening signals after differ heat process, especially to popcorn. Analysis of rusults to maize feeds, three kinds of feed samples that all of them contain transgene ratio(F1=1.431, F2=1.254, F3=1.186) more higher than 5% MON810 standard (S=0.998). So, it could show that three feeds contain high GM maize content. And three Bt maize from F1 feed be detected by restriction enzyme reaction and simplex PCR methologies with three event-specific primers, HS01/ cry- CR01, CDPK-cry03/CDPK-cry04 and Bt11 1-5’/cry1A 1-3’. According to above results, the systems for qualitative and semi-quantitative detection of GM Bt maize were acceptable.