Event-specific quantitative detection of genetically modified wheat B72-8-11 based on the 3′ flanking sequence

Abstract

Various PCR technologies have been developed for the execution of genetically modified organism (GMO) labeling policies of which an event-specific PCR detection method based on the flanking sequence of exogenous inserted DNA is the key trend in GMO detection due to its high specificity. In this study, 3′ flanking sequence between the host wheat DNA and the exogenous integrated gene construct of pHMW1Dx5 vector in transgenic wheat B72-8-11 was revealed by means of adaptor PCR, thus the fragment with the length of 210 bp was obtained, including a 144 bp unknown wheat genome DNA sequence. The event-specific quantitative PCR primers were designed based upon the revealed 3′ flanking sequence, and a SYBR Green I real-time PCR assay was subsequently applied. In the quantitative SYBR Green I real-time PCR assay, the LOD and LOQ were 10 and 20 haploid genome copies, respectively. In addition, three mixed wheat B72-8-11 samples with known contents were detected using this established quantitative PCR system, and the ideal results indicated that the developed event-specific quantitative PCR detection method could be used for identification and quantification of B72-8-11 wheat and its derivates.