Reliable Detection and Identification of Genetically Modified Maize, Soybean, and Canola by Multiplex PCR Analysis

Abstract

Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.

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

“…The main advantage of application of mPCR in this type of analysis is reduction of sample loads, time and resources: the developed method allows simultaneous amplification of several targets in one reaction. Similar detection/identification systems have been developed previously for a number of GMOs (James et al, 2003;Germini et al, 2004;Forte et al, 2005;Kim et al, 2007;Demeke and Ratnayaka, 2008). Moreover, the highly specific event-specific reaction, which follows the gene-specific reaction, confirms the presence and allows identification of a particular GMO in the tested sample, which was demonstrated in this study.…”

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

“…And most of multiplex detection assays have been performed using conventional PCR [22] [23]. In multiplex qPCR, you need to add different sets of primers and probes in order to detect different targets in one reaction.…”

Adventitious presence of GMOs: Scientific overview for Canadian grains

“…Therefore, development of multiplex PCR requires careful testing and validation. Nonetheless, multiplex qualitative PCR assays have been developed for the simultaneous detection of multiple GM events in canola (Demeke et al 2002;James et al 2003;Middleton and Stone 2003), soybean (Permingeat et al 2002;James et al 2003) and corn (Matsuoka et al 2001.…”

“…The international debate about AP has increased in breadth and depth because of global expansion in the development and cultivation of GM grains (James 2004;Runge and Ryan 2004;Cohen 2005). By 2004, 17 countries had commercialized production of GM cultivars of soybean, corn, cotton or canola on an estimated 81 million ha (James 2004).…”

“…By 2004, 17 countries had commercialized production of GM cultivars of soybean, corn, cotton or canola on an estimated 81 million ha (James 2004). According to Runge and Ryan (2004), some 63 countries now carry out research and development on more than 50 crop species, including all of the significant grain and oilseed commodity crops.…”