Development and Validation of Duplex, Triplex, and Pentaplex Real-Time PCR Screening Assays for the Detection of Genetically Modified Organisms in Food and Feed

Abstract

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

Detection of genetically modified maize in processed products, dry grains, and corn ears intended for fresh consumption in South Brazil

“…Two GM crop cultivars carrying the VT PRO event (BM 915 PRO and SHS 7920 PRO) presented positive results for all three targets. Furthermore, the other conventional and GM maize cultivars presented results that were expected based on register information, as demonstrated in previous studies (Reiting et al, 2007;Waiblinger et al, 2008;Dinon et al, 2011;Huber et al, 2013).…”

“…Although requiring special reagents and equipment, PCR can detect the presence of specific exogenous genes inserted into the plant genome (including the seeds), and represents the most widely used method to detect GM food and feed (Dinon et al, 2011;Branquinho et al, 2013;Cantelmo et al, 2013). In the present study, we used real time PCR to detect three GM-specific fragments: the promoter region p-35S derived from the cauliflower mosaic virus (Waiblinger et al, 2008;Huber et al, 2013), the terminator region, t-Nos, derived from the nopaline synthase gene of Agrobacterium tumefaciens (Reiting et al, 2007;Huber et al, 2013), and the main cry1A.105 gene from Bt (Dinon et al, 2011). These PCR assays were successfully implemented, in addition to an assay targeting the constitutive hmg gene, which was used as an endogenous control (Corbisier et al, 2010).…”

New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

“…In some cases, multiplex qPCR assays have been established to decrease the number of reactions to be performed (e.g. [19][20][21]). However, it should be noted that multiplexing using qPCR has its limitations [22], i.e.…”

Screening for six genetically modified soybean lines by an event-specific multiplex PCR method: Collaborative trial validation of a novel approach for GMO detection

“…The asymmetric LOD (LOD asym ) should be determined for multiplex qualitative PCR modules according to the ENGL guidance document (ENGL 2015). It is defined as a performance parameter for the sensitivity of a multiplex assay when one target is present at very low concentration in comparison with the other targets at high concentration (Huber et al 2013;Broeders et al 2014). However, for the multiplex assay the competitive effects between target amplifications are not relevant, because any positive PCR signal must be verified by singleplex identification tests for all respective targets.…”

“…Validated duplex, triplex and pentaplex assays combining element-and constructspecific real-time PCR methods are available and allow time-and cost-reduced GMO screening (Waiblinger et al 2008;Bahrdt et al 2010;Dorries et al 2010;Huber et al 2013). These multiplex TaqMan PCR assays are based on probes labelled by up to five different fluorescent dyes for simultaneous detection of the different target sequences.…”