Event-Specific Qualitative and Quantitative Polymerase Chain Reaction Analysis for Genetically Modified Canola T45

Abstract

Polymerase chain reaction (PCR) methods have been the main technical support for the detection of genetically modified organisms (GMOs). To date, GMO-specific PCR detection strategies have been developed basically at four different levels, such as screening-, gene-, construct-, and event-specific detection methods. Event-specific PCR detection method is the primary trend in GMO detection because of its high specificity based on the flanking sequence of exogenous integrant. GM canola, event T45, with tolerance to glufosinate ammonium is one of the commercial genetically modified (GM) canola events approved in China. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of T45 canola was cloned and revealed by means of TAIL-PCR. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence, and qualitative and quantitative TaqMan real-time PCR detection assays employing these primers and probe were developed. In qualitative PCR, the limit of detection (LOD) was 0.1% for T45 canola in 100 ng of genomic DNA. The quantitative PCR assay showed limits of detection and quantification (LOD and LOQ) of 5 and 50 haploid genome copies, respectively. In addition, three mixed canola samples with known GM contents were detected employing the developed real-time PCR assay, and expected results were obtained. These results indicated that the developed event-specific PCR methods can be used for identification and quantification of T45 canola and its derivates.

Implementation of monitoring for genetically modified rapeseed in Serbia

“…All GM-positive samples were analyzed using the event specific primers for T45, RF3 and RT73 (Table 1). As expected, in the established PCR assay, the primers specific for T45 oilseed rape (Yang et al 2006) produced 233 bp DNA fragment in T45 positive controls (Figure 2, lines 3 and 4) and no amplification in non-transgenic rapeseed (Figure 2, line 5). All samples were analysed in triplicate.…”

Development of a Multiplex Ligation Dependent Probe Amplification (MLPA) Module for Simultaneous Detection of Five Genetically Modified Rapeseed Events

“…Therefore, the PCR efficiency is equal or very similar and interactions between the primers can be excluded. The obtained detection limits of our developed MLPA assay are in the range of a rapeseed qPCR [24] [25]. One should keep in mind that we used genomic DNA as starting material, whereas plasmids are often used as calibrators in qPCR [26].…”

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

“…Finally, to overcome this problem, event-specific assays have been developed, which are the most specific reactions for identification of GMOs. They target a unique site comprising a junction between the transgenic insert and the host genome (Holst-Jensen et al, 2003;Yang et al, 2006;Marmiroli et al, 2008;Dörries et al, 2010). Different varieties of polymerase chain reactions are commonly used for monitoring of GMOs in foods and animal feeds (Shin et al, 2013;Kim et al, 2014;Meriç et al, 2014;Datukishvili et al, 2015;Turkec et al, 2016).…”