Use of pJANUS™-02-001 as a calibrator plasmid for Roundup Ready soybean event GTS-40-3-2 detection: an interlaboratory trial assessment

Abstract

Owing to the labelling requirements of food and feed products containing materials derived from genetically modified organisms, quantitative detection methods have to be developed for this purpose, including the necessary certified reference materials and calibrator standards. To date, for most genetically modified organisms authorized in the European Union, certified reference materials derived from seed powders are being developed. Here, an assessment has been made on the feasibility of using plasmid DNA as an alternative calibrator for the quantitative detection of genetically modified organisms. For this, a dual-target plasmid, designated as pJANUS™-02-001, comprising part of a junction region of genetically modified soybean event GTS-40-3-2 and the endogenous soybean-specific lectin gene was constructed. The dynamic range, efficiency and limit of detection for the soybean event GTS-40-3-2 real-time quantitative polymerase chain reaction (Q-PCR) system described by Terry et al. (J AOAC Int 85(4):938–944, 2002) were shown to be similar for in house produced homozygous genomic DNA from leaf tissue of soybean event GTS-40-3-2 and for plasmid pJANUS™-02-001 DNA backgrounds. The performance of this real-time Q-PCR system using both types of DNA templates as calibrator standards in quantitative DNA analysis was further assessed in an interlaboratory trial. Statistical analysis and fuzzy-logic-based interpretation were performed on critical method parameters (as defined by the European Network of GMO Laboratories and the Community Reference Laboratory for GM Food and Feed guidelines) and demonstrated that the plasmid pJANUS™-02-001 DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products.

Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms

“…Moreover, three parameters were evaluated to check that the PCR amplification kinetics in the sample extract was not significantly different from those of the calibrant. Previous publications [8,12,29] have already addressed some of these issues, also regarding the variations of the between-laboratory performance. However, they were limited to one specific GM event.…”

“…The use of plasmids which contain cloned junctionspecific and endogenous sequences-so-called dual-target plasmids-as calibrants for GM quantification has been demonstrated previously [3][4][5][6][7][8][9][10][11]. However, the validity of the conclusions drawn from these studies is limited, as they have been based on repeatability studies within the same laboratory.…”

A Particular Case of Novel Food

“…Quantification based in the direct comparison of results obtained from processed samples and those from CRMs should be taken with care, because matrix effects and the loss of integrity of the DNA molecules during processing could bias the estimation of the actual GMO content of the samples. Novel synthetic DNA standards, including genomic and plasmidic DNA molecules that contain the event-specific and reference sequences of interest, have been also proposed as calibrators in real-time PCR methods for GMO quantification [95][96][97][98][99] .…”

New plasmid calibrators for geminivirus-resistant (EMB-PV051-1 event) common bean (Phaseolus vulgaris L.) quantitation using simplex and duplex qPCR

“…pDNA calibrators have some advantages compared to gDNA: they are easier to construct; can be produced at lower cost; they are stable frozen or lyophilized and can be stored for a long period of time; they are well-defined and have a fully characterized sequence (Burns et al, 2006;Caprioara-Buda et al, 2012;Wang et al, 2011). Plasmid calibrators have been considered adequate CRM because of their convenience and lower cost (Lievens et al, 2010;Meng et al, 2012).…”