Event-specific qualitative and quantitative polymerase chain reaction methods for detection of insect-resistant genetically modified Chinese cabbage based on the 3'-junction of the insertion site.

摘要

Transgenic Chinese cabbage 416-3 was developed in Korea by a transformation event involving a modified insect resistant gene (cry1Ac1). To monitor unintended release of GM Chinese cabbage in the future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of GM cabbage is requisite. To develop qualitative and quantitative PCR methods for the insect-resistant GM Chinese cabbage, a cytosolic glutathione reductase (BcgGR1) gene was used as the endogenous reference gene. Primer pairs CGR-1/-2, amplifying the Chinese cabbage endogenous gene, yielded an expected amplicon of 121 bp, whereas no amplified product was observed when DNA samples from 7 non-cabbage plants were used as templates. The event-specific primer pairs amplifying the junction site between the endogenous genome sequence and the transferred DNA of GM event 416-3, produced amplicons of desired size by qualitative PCR assay. An event specific quantitative PCR detection method was established using a TaqMan probe and a standard plasmid as a reference molecule, which contained both endogenous and event-specific sequences. For the validation of this method, 3 different compositions of w/w mixed samples (containing transgenic DNA at 5, 1 and 0.5% of total DNA in the control samples) were quantified. Precision, expressed as standard deviation (SD) and relative standard deviations (RSD), deviated by 0.03-0.26% and 4.75-8.06%, respectively. These results clearly demonstrate that the PCR methods developed herein can be used for event-specific qualitative and quantitative testings of insect-resistant GM Chinese cabbage.