Comparison and transfer testing of multiplex ligation detection methods for GM plants

Abstract

BackgroundWith the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.ResultsOf the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).ConclusionsFrom the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.

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

“…the number of dyes that can currently be detected, the occurrence of false positives when the targets to be multiplexed become too numerous and/ or may interact and the loss of sensitivity. Furthermore, high-throughput technologies, as, for instance, NASBA implemented microarray analysis (NAIMA) [23,24], microdroplet PCR implemented capillary gel electrophoresis (MPIC) [25], multiplex ligation detection methods [26] (and references therein), a combined microchip-PCR and microarray system (MACRO) [27], digital PCR [28,29] and next-generation sequencing (NGS) [30][31][32], have been developed and their possible use in GMO detection was demonstrated. These methods are, however, still too costly and/or cumbersome for routine use and often require expensive equipment and/or specialised data analysis tools and staff.…”

Inter-laboratory Testing of GMO Detection by Combinatory SYBR®Green PCR Screening (CoSYPS)

“…In most cases, GMO screening approaches apply quantitative PCR (qPCR) methods for detecting the presence of GM material in food and feed samples. More complex strategies employing microarrays (e.g., GMOchips (Leimanis et al 2006), NAIMA (Morisset et al 2008), and PADLOCK probes ligation (Ujhelyi et al 2012)) have been attempted, but are, however, still under validation (NAIMA, padlock approach) or received limited distribution within the enforcement world (GMOchips). Thus, today, qPCR screening is by far the most commonly applied approach in screening for GM materials in food and feed products.…”

Next generation DNA sequencing based strategies; towards a new era for the traceability of endangered species and genetically modified organisms

“…By screening for the presence or absence of specific GMO elements, the presence of elements incompatible with the presence of only authorised GMOs may indicate the presence of one or multiple UGMOs. Subsequent sequencing of those unexplained GMO targets and their flanking regions might identify the UGMOs present in the sample containing the unexplained GMO elements [216,218,219]. The obtained sequence information can be compared to the reference GMO sequences for identification [220].…”