The adoption of crops that are genetically modified organisms (GMOs) has continuously increased over the last decade with 148 million hectares grown in 2010 worldwide [1
]. Because of the increasing number of GM crops, the analysis of an individual food or feed sample for the potential presence of GMOs becomes more complex, time-consuming and expensive. To overcome these problems it is necessary to develop a method which can identify many GMO-derived DNA targets in a single experiment, at a sensitive level, reducing both cost and analysis time. The potential presence of unauthorized GM crops makes the situation even more complicated [2
Currently, the most common method to detect and identify GMOs in food and feed products is real-time polymerase chain reaction (PCR). For most targets this method has a limit of detection (LOD) of 0.1% or less. In the scientific literature, different multiplex GMO detection methods have been described but various problems with detection level and specificity have been reported. Ligation-based systems seem very promising approaches to detect GMOs in a multiplex setting in a sensitive and specific way.
Ligation was one of the first tools in the hands of molecular biologists for cloning and DNA manipulation and has played a major role in explanation of gene functions. It was also found that ligation can be used for detection of specific DNA sequences [4
]. During the 1990s several ideas and theories were examined for making ligation detection more sensitive and applicable for multiplex detection. One of the resulting strategies used so-called padlock probes (PLPs). PLPs were designed to be linear with the ligation sites at the extremities. The PLP was shown to be circularized after ligation [5
] and with this method up to 10,000 DNA targets were detected simultaneously in a human setting [6
]. In the area of single-nucleotide polymorphism (SNP) detection of up to thousands of targets has been reached [7
]. A PLP usually contains universal primer sites for PCR amplification and a universal microarray can be used for detection and identification (Figure ). Such a padlock system was adapted to detect and identify (GMO) crops [8
Figure 1 Scheme of the padlock ligation detection procedure. A mix of linear padlock probes can hybridize to their genomic counterparts, after which the juxtaposed ends are ligated to form a circular molecule. Only ligated, circular molecules are amplified by (more ...)
In a tenplex PLP experiment different genomic targets in GTS 40-3-2 soy, MON1445 cotton and Bt176 maize were detected down to at least 1% [8
]. The PLP system can be used not just for GMO detection but also for other nucleic acid experiments. It was for instance used for SNP-based genotyping in allohexaploid wheat [10
Other ligation based techniques have been developed to detect GMOs as well. One of these uses two separate " bipartite ligation" probes for each target. After the amplification of the targets the detection can be performed either by capillary electrophoresis or by microarray hybridization. This kind of ligation-dependent probe amplification (LPA) system was used by Moreano et al. [11
] to detect several targets. In their study two endogenous targets and two event specific junction regions were detected simultaneously. GMO maize DNA (0.1%) was detected in the presence of 5% GM soy DNA and vice versa. This group improved the above-mentioned LPA technique for more targets [12
]. This LPA technique was also used for simultaneous detection of 10 allergens [13
]. Holck et al. [14
] developed a nineplex ligation-dependent probe amplification method for detection of seven GM maize events, one GM maize construct and one endogenous maize reference gene.
A so-called SNPlex method, which used also two separate probes, has also been tested for GM detection by Chaouachi et al. [15
]. Probes in this paper contained universal primer sites for the PCR and specific ZIP-codes (ZIPChute probe). As one of the primers was biotinylated, the biotinylated amplicon was captured onto a streptavidin coated surface after the PCR. These ZIPs contained a unique sequence that enabled their size differentiation during electrophoresis. This assay allowed the simultaneous detection of potentially up to 48 DNA sequences (endogenous, element-, construct-, and event-specific targets). In their paper simultaneous detection for up to seven targets was shown with a detection limit range of 0.1-1%.
Peano et al. [16
] also applied separate probes and the ligation detection reaction was combined with a universal array approach. They performed an extra pre-amplification step before the ligation and describe the detection of five different GMOs when present at 0.4% each, relative to non-GM (conventional) material.
Ligation based systems have been used for other nucleic acid experiments as well. Ericsson et al. [17
] used a dual-tag microarray platform for high-performance nucleic acid analysis. After the dual-tag probe ligation, solution phase rolling circle amplification was performed and the detection was carried out on chip. Different other multiplex approaches have been described by several authors [18
] to detect GMOs but none of the techniques have so far shown more multiplicity than the ligation detection methods.
It was noted that the above-mentioned ligation based methods used various ligation protocols, which are radically different from each other with regard to temperatures, incubation times and number of reaction cycles (Figure ). There are also some differences among the types of probes, probe concentrations and the type of ligation enzymes, but all publications seem to reach similar sensitivities so far. In the literature different kinds of PCR parameters have been described as well, but the different PCR parameters are not likely to have so much effect on the sensitivity compared to the ligation step. The large differences among these ligation procedures led us to compare different protocols in a common sample setting to try and find the factors that are most important for specific and sensitive GMO (multi)detection.
Figure 2 Differences among the published ligation protocols. For each method, a temperature (y-axis) × time diagram (x-axis) is shown as it was used in the indicated references. LPA: Ligation-dependent Probe Amplification, DTM: Dual Tag Microarray, PPLMD: (more ...)
To this end the present study aimed to compare different ligation protocols including reaction temperatures with the PLP system in different GM mixes. Three of the selected ligation protocols were GMO detection related [8
] and the other two were used for other types of nucleic acid analysis [10
]. The detection was performed with TaqMan probes designed for PLPs in all cases, using the same real-time PCR parameters in fourplex and the best results were confirmed on microarray. Further aim was to test the specificity of the system using the best ligation protocol, based on the results of the ligation comparison. Finally, the transferability of method was tested in an interlaboratory exchange study as an initial validation step of the approach.