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

Abstract

specificity and repeatability were assessed as well as the robustness of the methods. Additionally, the reproducibility was evaluated by transferring the methods to a second laboratory. Both methods allow a specific, sensitive and repeatable detection of the respective targets in food and feed samples and can be easily applied in a routine laboratory. Moreover, they can be used together with previously validated SYBR ® Green qPCR methods to expand the panel of screening methods. This allows an extended coverage of the GM events authorised in the EU and adds discriminative power to the screening phase.

Semiautomated TaqMan PCR screening of GMO labelled samples for (unauthorised) GMOs

“…The P-35S [26] was not detected by us in DP98140 maize but was reported as detected by Block et al [20] The DP98140 maize contains no P-35S according to the EUginius database. T-35S [17] was found to be positive in T45 canola and DAS59122 maize and reported as unexpectedly positive by Block et al According to the EUginius database both GMOs contain a T-35S so a positive signal can be expected in T45 canola and DAS59122 maize.…”

Current and New Approaches in GMO Detection: Challenges and Solutions

“…In addition, some markers more discriminative, such as Cry3Bb, gat-tpinII, and t35S pCAMBIA, and taxon-specific markers could also be used. This step allows establishing a list of the potential GMO present in the tested samples and preventing further unnecessary assays in the subsequent steps ( Table 2 ) [ 28 , 30 – 34 ]. Several of these screening markers are validated, based on minimum performance requirements, at the EU level following ring trials and are included in the Compendium of reference methods for GMO analysis [ 35 ].…”

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

“…Settings for the amplicons were Ta = 60 °C ± 5 °C; amplicon length 60-120 bp. The detection method was partially based on the SYBR Green qPCR CoSYPS method described by Broeders et al [10]. Primers and probes were synthesised by Biolegio (Nijmegen, Netherlands) with a high-purity method.…”

“…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]. Detection and identification of GMOs is not static.…”