Development of a polymerase chain reaction assay for detection of three canola transgenes

Abstract

Many countries are developing or implementing regulatory requirements to monitor for the presence of genetically modified (GM) materials in seeds, grain, and derived food products using DNA and protein-based methods. There is no published report on the detection of different GM transgenes in canola, and this study is aimed at developing qualitative PCR methods for the three major GM transgenes commercially available in canola. Primer sequences were generated from GenBank and previously published information to develop a polymerase chain reaction (PCR) detection method for Roundup Ready (glyphosate tolerance, GT73 event), Liberty Link (glufosinate ammonium tolerance, HCN92 event), and BX (Bromoxynil tolerance, OXY235 event) canola varieties. On using PCR, two primer pairs for each of the GT73 and HCN92 and one primer pair for OXY235 amplified specific amplicons for the three GM transgenes. All three GM transgenes were detected simultaneously by multiplexing the five primer pairs in a single PCR reaction. Multiplexing of the five primer pairs for DNA prepared at 1% (one GM seed in 99 non-GM seeds) and 0.5% (one GM seed in 199 non-GM seeds) levels generated the expected DNA fragments for GT73, HCN92, and OXY235. This information will lay the groundwork for the development of a quantitative PCR assay for canola transgenic events.

Multiplex PCR assays for qualitative detection and identification of the GT73, Ms8, Rf3 and T45 varieties of genetically modified oilseed rape

“…For the eventspecific reaction, the LOD value was determined at 0.01% (w/w) for the GT73, Ms8 and Rf3 rape lines, and 0.025% (w/w) for T45 rape ( Figure 6B). Both developed assays displayed higher sensitivity than a similar multiplex PCR-based reaction dedicated for GM rape detection as previously stated by Demeke et al (2002) and Kim et al (2007). It should be noticed that the obtained LOD levels were absolutely optimal for the multiplex PCR method used for GMO content monitoring in feedstuffs in Poland.…”

Multiplex qualitative PCR assay for identification of genetically modified canola events and real-time event-specific PCR assay for quantification of the GT73 canola event

“…Qualitative PCR detection methods have been published for GM canola events (Demeke, Giroux, Reitmeier, & Simon, 2002;James, Schmidt, Wall, Green, & Masri, 2003;Middleton & Stone, 2003). Many of the PCR primers used in these published methods are construct-specific or derived from cauliflower mosaic virus promoter and p-NOS terminator and none of them are event-specific.…”

Development of a multiplex event-specific PCR assay for detection of genetically modified rice

“…Lane M: marker (50 bp DNA Ladder); Lanes 1-7: 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, and 0.01% of a mixture of GM rice varieties (TT51-1, KMD and KF6); Lane 8: non-GM rice; Lane 9: no template variant of PCR method, multiplex PCR method can be simultaneously performed of multiple event-specific sequences with more than one pair of primer sets in single-tube reactions and considered time-saving, low-cost and effective, as compared to the standard simplex PCR systems (Shrestha et al 2010;You 2014). Multiplex PCR assays of different GMPs have been developed, such as GM soybean (Kim et al 2009;Kim et al 2013), GM cotton (Demeke et al 2002;Kim et al 2008;Yang et al 2005), GM canola (Kim et al 2007), and GM maize . In this study, multiplex PCR assay was developed for the qualitative detection of three GM rice genes amplified concurrently in a single PCR tube.…”