Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms

Abstract

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-4875-9) contains supplementary material, which is available to authorized users.

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).…”

“…All of these GM events carry at least one promoter and one terminator region, in addition to the main gene (cry, vip, epsps, etc.). The promoter region p-35S (from the cauliflower mosaic virus) and the terminator t-Nos (from the nopaline synthase gene) are the most commonly used in GM maize seeds (Wolf et al, 2000;Dinon et al, 2011).…”

Multiplex PCR system to track authorized and unauthorized genetically modified soybean events in food and feed

“…D€ orries, Remus, Gr€ onewald, Gr€ onewald, and Berghof-J€ ager (2010) developed a multiplex real-time PCR kit to detect P-35S, T-nos, P-FMV, and the bar gene for GMO screening purposes. Grohmann, BrunenNieweler, Nemeth, and Waiblinger (2009) and Dinon et al (2011) reported screening methods that targeted cording genes with herbicide tolerance and insect resistance. Randhawa, Chhabra, and Singh (2009) established a multiplex PCR system that targeted selectable markers and reporter genes for GMO screening.…”

Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

“…Therefore, additional detection methods are sometimes necessary in order to target specific (groups of) GMOs. Some recent examples of additional qPCR screening methods are the element bar and construct ctp2/cp4epsps [21], the elements cry1A.105 and cry2Ab2 [15], promoters P-FMV, P-nos, P-SSuAra, P-TA29, P-ubi, P-rice actin and terminators T-35S, T-E9, T-ocs, T-g7 [14], element vip3A [29], and element cry1Ab/Ac and construct P-ubi/cry [22]. Recent examples of SYBR ® Green detection methods are P-nos and P-FMV [9], cry3Bb and gat/T-pinII [10], and the application of Combinatory SYBR ® Green PCR Screening (CoSYPS) [4,30,42].…”