Event-specific Qualitative Polymerase Chain Reaction Research Articles

Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange Petunia

In 2017, various orange coloured petunia on the market turned out to be genetically modified (GM) without an official authorization for commercialization. Sequence analysis suggested these undeclared plants most probably originated from a plant transformation experiment performed in the 1980s. For a deeper understanding how GM petunia entered classical breeding programmes worldwide, and whether they originated from a single source or not, we undertook a molecular genetic characterization of the T-DNA integration sites in different GM petunia cultivars and breeding lines. By means of genome walking, we isolated different T-DNA sequences, which are located at the junctions between the T-DNA(s) and the petunia DNA. Based on the results obtained we conclude that there are at least two T-DNA copies of different lengths. This is supported by Southern blot analysis. For T-DNA1, the 3′-junction sequence was isolated, whereas the 5′-junction remained unclear. In contrast, for T-DNA2, the 5′-junction sequence was isolated, whereas the sequence isolated from the 3′-region consists only of T-DNA, but did not include the junction from the T-DNA to the petunia DNA. We developed primers for event-specific PCRs and screened a set of three orange GM petunia cultivars and 126 GM offspring from a commercial breeding program. We show that both T-DNA copies are present in all our tested GM petunia samples, which underpins the assumption of a single transgenic origin of the undeclared GM petunia. Most likely, the two T-DNAs are integrated in close proximity into the petunia genome.

Multiplex event-specific qualitative polymerase chain reaction for detecting three transgenic rice lines and application of a standard plasmid as a quantitative reference molecule

The three most well-known genetically modified (GM) rice lines in China are TT51-1, KMD1, and KF6. The purposes of this study were to establish a multiplex event-specific qualitative polymerase chain reaction (meqPCR) system for simultaneous detection of the three transgenic rice events and to construct a plasmid as the reference molecule for quantitative analysis. Event-specific primers for each event were selected or designed by focusing on the transgene borders between the inserted DNA and the flanking rice DNA. The developed meqPCR was anticipated to detect distinct amplicons as 454, 398, 301, and 250bp from KF6, KMD1, TT51-1, and the rice endogenous reference gene, respectively. The robustness of the meqPCR was tested with different levels of the three transgenic rice genomic DNAs, and the sensitivity threshold of the meqPCR was at least 50ng of 0.1% rice DNA for each event when the three transgenic rice events present and with other GM materials together. The constructed plasmid was evaluated using mixed samples with known GM contents in real-time quantitative PCR. The results indicated that the constructed plasmid was acceptable and suitable for GM rice quantitative analysis.

Event-Specific Qualitative and Quantitative Polymerase Chain Reaction Methods for Detection of Genetically Modified Rapeseed Ms8Rf3 Based on the Right Border Junctions

Ms8xRf3 is a genetically modified rapeseed hybrid which is widely cultivated in Canada and exported to some other countries for production of foodstuffs or fodder. In this study, the genomic sequences flanking the right borders of the integrated transgenic sequences in the Ms8xRf3 genome were characterized and showed high similarities with the bacterial artificial chromosome clone of Chinese cabbage. Event-specific qualitative polymerase chain reaction (PCR) methods were established with the primers and probes targeting the junction regions to produce a 123 base pair (bp) product for the Ms8 event and 92 bp for the Rf3 event. The absolute detection limit of qualitative PCR was 2.5 initial template copies for the Ms8 event and 50 copies for the Rf3 event. Quantitiative detection methods were established, with the absolute quantification limit being approximately 25 initial template copies.