Event-specific detection of genetically modified wheat B73-6-1 based on the 3′-flanking sequence

• Published in: European food research & technology Vol. 235; no. 6; pp. 1149 - 1159
• Main Authors: Zhang, Minghui, Huo, Nan, Liu, Ying, Qiu, Youwen, Ao, Jinxia, Luan, Fengxia, Li, Qingzhang, Gao, Xuejun
• Format: Journal Article
• Language: English; German
• Published: Berlin/Heidelberg Springer-Verlag 01.12.2012; Springer; Springer Nature B.V
• Subjects: Agriculture; Analytical Chemistry; Biological and medical sciences; Biotechnology; Cereal and baking product industries; Chemistry; Chemistry and Materials Science; Corn; Deoxyribonucleic acid; DNA; DNA polymerase; Food; Food industries; Food Science; Forestry; Fundamental and applied biological sciences. Psychology; General aspects; Genes; Genetic testing; Genetically altered foods; Genomes; Genomics; Methods of analysis, processing and quality control, regulation, standards; Original Paper; Quarantine; Rice; Seeds; Studies; Triticum aestivum; Wheat; Transgenic wheat B73-6-1; Genetically modified organisms (GMOs); Flanking sequence; Genome walking; Event-specific detection; Analysis method; Cereal; Control method; Detection; Genome; Genetically modified organism; Wheat
• ISSN: 1438-2377; 1438-2385
• DOI: 10.1007/s00217-012-1848-y

In this study, 3′-flanking sequence between the host plant DNA and the integrated gene construct of pHMW1Dx5 vector in transgenic wheat B73-6-1 was revealed by means of adaptor PCR; thus, the fragment with the length of 3.1 kb was obtained, including a 190-bp wheat genomic DNA, which demonstrates that this HMW-GS gene was located on the wheat chromosome 3B. And the event-specific PCR primers were designed based upon the revealed 3′-flanking sequence; the conventional qualitative PCR and quantitative SYBR real-time PCR detection methods employing these primers were successfully developed. In conventional qualitative PCR assay, the limit of detection was 0.1 % for B73-6-1 wheat genomic DNA for one reaction. In the quantitative SYBR real-time PCR assay, the limit of detection and limit of quantification were 10 and 100 haploid genome copies, respectively. In addition, three mixed blind wheat samples with known B73-6-1 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event-specific real-time PCR detection systems were reliable, sensitive and accurate.