Development of PCR methods for detection and quantification of genetically modified maize

Jabbari Farhoud, Houman
This study describes development of methods for screening, identification and quantification of genetic modifications in maize samples. Totally 88 maize samples were collected randomly throughout Turkey in three years from 2006 to 2008 and were analyzed. Two maize samples that were detected as GM positive in previous studies were selected as positive controls. Following the DNA extraction by manual CTAB method, conventional PCR methods were employed for screening of genetic modifications in samples by detecting of P-35S and T-NOS. Qualitative PCR methods were conducted for target specific detection of cry and pat genes. Construct-specific and event-specific PCR assays were designed for detection of Bt11, Bt10 and Mon810 maize events. Specific primers and corresponding probes labeled with reporter and quencher dyes were designed for both absolute and relative quantification of Bt11 and Mon810 in samples by using TaqMan probe method. Comparing the absolute and relative quantification results indicates that there is correlation between them. In order to verify the accuracy of the quantification methods, three parallel applications were conducted according to the CRL validated protocol. The statistical analyses were performed to check the precision and repeatability of the quantification experiments by in-house validation methods. Regarding the Repeatability Relative Standard Deviation (RSDr) values of absolute and relative quantifications of Bt11 and Mon810 systems majority of the validation results accomplish the ENGL requirements for quantification of GMOs. According to screening assays, the overall results indicate that five samples (H3, H48, H73, 4M, 4G) were detected as GM positive. While the samples H3 and H48 were identified as Bt11, it was shown that the sample 4M and 4G contains both of the Bt11 and Mon810 maize events. Bt11 quantification results show samples H3 and 4G respectively with 1.06% and 5.36% exceed the 0.9% threshold level. Amount of Mon810 in samples was determined as 1.33 % for 4M and 17.32% for 4G which is higher than 0.9% threshold level. Sample H73 which was detected as GM positive did not contain Bt11 and Mon810 maize events. Since the methods developed in this study reduce dependence on commercial kits they would contribute to expansion of GMO testing in Turkey with lower cost. However the methods developed in this work should be extended to other maize events and their validation procedure should be completed.


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Citation Formats
H. Jabbari Farhoud, “Development of PCR methods for detection and quantification of genetically modified maize,” Ph.D. - Doctoral Program, Middle East Technical University, 2010.