Development of a Novel Detection Method Targeting an Ultrashort 25 bp Sequence Found in Agrobacterium-Mediated...

Agrobacterium-mediated transformation is the most commonly used technique for plant genetic engineering. During the transformation, a T-DNA region, which is flanked by the right border (RB) and the left border, is transferred to plant nuclear chromosomes. Simultaneously, a sequence adjacent to the RB on T-DNA is frequently transferred to plant genomes together with the intentionally introduced recombinant DNA. We developed a novel polymerase chain reaction (PCR)-mediated detection method targeting this region. The conserved sequence of the region found in genetically modified (GM) crops is only 25 bp in length. To detect this ultrashort 25 bp sequence near the RB region, we designed a primer set consisting of a 12-base forward primer and a 13-base reverse primer. The predicted band was detected from GM crops by optimizing the PCR conditions. We used lateral flow DNA chromatography for rapid and inexpensive detection. The developed method would be applicable for screening the GM crops generated by Agrobacterium-mediated transformation.