NRL-GMO GMODetec research project ( )

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1 NRL-GMO GMODetec research project ( ) Authors: IPH: Barbau-Piednoir, E., Lievens, A., Leunda Casi, A., Roosens, N., Van den Bulcke, M., Sneyers, M. CRA-W: Debode, F., Jansens, E., Berben, G. ILVO: Ruttink, T., Taverniers, I., De Loose, M. The GMODetec project (RT-06/6: Ontwikkeling van een algemene strategie voor detectie, identificatie en kwantificering van genetisch gemodificeerd materiaal in voedingsproducten en veevoeder ) is a collaborative project between the three labs of the NRL-GMO consortium (IPH, CRA-W and ILVO). The project is funded by the Federal Public Service (FPS) for Health, Food chain safety and Environment and coordinated by IPH. The overall objective of the project is to develop integrated strategies and models for detection, identification and quantification of GMOs in food and feed. The project focused on development of: a strategy allowing development of a GMO detection model for all GM-events where sufficient information (especially on the DNA sequences) is available. The aim here is to develop PCR methods based on the comparative analysis of the available official DNA sequences. The analytical results will be integrated in a mathematical decision model allowing the identification of the transgenic material present in the product. a strategy allowing the development of a GMO passport of a product by means of the methods employed in the total genome analysis of an organism. By suitable choice of the reference points an image of all the transgenic sequences present in the product will be generated. Further, the mathematical decision model for the authorized GMOs has to be extended to a model allowing the visualisation of the GM-composition of the DNA extracted from a product. In this case the presence of non-authorized GMOs can be traced by comparative analysis of the generated fingerprints with the transgenic sequences present in authorized GMOs. The GMO passport strategy allows the development of a general GMO detection model in a uniform manner. This model will be less dependent on the available information of the respective GMOs and will be flexible by the reference points that have to be taken in the analysis. I. Development of qpcr methods - Screening A. CoSYPS (IPH) During the three year GMODetec Project, ISP worked on developing and improving CoSYPS (Combinatory SYBR Green qpcr Screening). This decision support system, patented in 2008, allows the identification of the potential presence of GMOs in food matrices 1,2. This screening method is flexible and can be adapted according to the requirements and the type of GMO being sought. At this moment in time, IPH has 14 SYBR Green qpcr screening methods (plants, soya, maize, oilseed rape, cotton, rice, beetroot, the 35S promoter and the nopaline synthase terminator 3, the CP4-EPSPS, PAT/pat and PAT/bar genes which confer tolerance to glyphosate (Roundup ) and glufosinate (Basta, Liberty..) herbicides respectively, the CryIAb gene which confers resistance to certain insects (lepidopteran pests) such as the European corn borer (Ostrinia nubilalis), and checking for the presence of Cauliflower Mosaic Virus : CaMV in the sample. (Checking for the presence of p35s positive due to CaMV). These real time PCR methods use SYBR Green technology. This is an intercalating agent which only fluoresces 14

2 when it is intercalated in double-stranded DNA ; this chemical agent allows the amplification of the DNAg matrix to be monitored at each PCR cycle 4. Figure 1: On the left: an example of amplification measured in Real Time PCR. Fluorescence is measured on the Y-axis, the PCR Cycle number on the abscissa. On the right: a Real Time PCR (ABI 7300) The analysis of the results obtained from the SYBR Green qpcr, based on a mathematical decision support system linked to CoSYPS, allows to establish a list of GMO events possibly present in the tested sample 2. During the second stage of the analysis, the presence of these GMO events will be tested through a transformation eventspecific TaqMan 4 method 5. As CoSYPS is a flexible system, other methods can be developed and added to the system in order to allow the detection of future GMOs; ISP is in fact already involved in a European project called GMOSeek ( ) in the course of which 5 new methods will be developed. During the GMODetec Project, a Single Target Plasmid (STP) was constructed for each of the methods developed. The use of a mixture of all these STPs was validated for being used as the sole positive control of all the screening methods. During the project s third year, CoSYPS was evaluated through an inter-laboratory test which 13 European laboratories took part in. The results of this inter-laboratory test revealed that CoSYPS could be transferred and used in other GMO detection analysis laboratories. We can conclude that CoSYPS is a versatile and operational screening method which can be transferred to other laboratories with a qpcr platform. The next step is to design and market ready-to-use plates (pre-filled and lyophilized) in order to simplify and reduce the cost of analysis preparation, thereby making this method accessible to as many laboratories as possible. 15

3 B. Development of new screening elements for GMO detection using TaqMan (CRA-W) GMO detection through screening using TaqMan methods was until recently almost solely based on searching for the 35S promoter and the NOS terminator The ever-growing list of GMOs authorized in food requires new screening markers to be developed. The GMODetec project allowed CRA-W to develop a significant number of screening methods based on TaqMan targeting: promoters: pfmv35s, price Actin, pssuara, pta29, pnos terminators : t35s, tocs, tg7 genes: gox, gus, EPSPS, bar, hsp70. II. Searching for unknown GMO events A. SELLUX (CRA-W) Technology Technology called SELLUX was implemented to try to amplify unknown sequences close to sequences amplified during screening and unexplained by known GMO events. SELLUX technology tries to amplify this area with the help of a standard length primer positioned on a known screening element and a short primer which must hybridize in a region whose sequence is unknown. Various types of technology were considered: the integration of LNA bases, the integration of inosine bases into different places, tests with different chemistries such as LUX primers, SYBR Green and TaqMan probes. Only the SYBR Green method showed compatibility between the use of short primers and obtaining Real Time PCR signals. The use of inosine bases also had a positive effect on the signals. The conclusion of this work is that amplification with short primers is possible but because of the lack of sensitivity generated by using short primers, it must be recognized that in the current state of knowledge, this technique is not yet suitable for creating profiles relating to the different genetically modified events which could be encountered. 5bp primer + inosin Reverse primer Unknown region Known region Figure 2: The experiments were conducted to find out if it was possible to generate an amplicon with very short primers likely to hybridize in a region whose sequence is unknown. Inosine bases (bases which can pair up indiscriminately with A, C, G or T) were used to increase the hybridization properties of short primers. 16

4 B. GGO passport (ILVO) The objectives of ILVO as a research partner within the GMODETEC project were bipartite: on the one hand the developing and further optimising of a protocol for anchor-pcr fingerprinting; on the other hand the developing of integrated strategies for determining the composition of a GGO sample, including the detection and characterising of unauthorised GGOs. The second objective is in keeping with the final objective of the project, namely the developing and optimising of a so-called GGO passport identification technology. The first objective within the project was the developing and further optimisation of a protocol for anchor-pcr fingerprinting. A new fluorescent anchor-pcr protocol was drawn up and published utilising anchor-primers for the screening elements p35s and t-nos 12. Therefore a perfect link is possible with the matrix screening approach, which, amongst other things, makes use of these elements. In the second project year this protocol was further refined and extensively tested on wild types as well as GGO materials. The second objective of ILVO concerned the elaboration of alternative strategies for the tracing of unauthorised GGOs. Strategy 1 is based on molecular analytic detection and requires knowledge of the GGO DNA sequences for the design of specific analytical tests, but does not call for a knowledge of the GGO composition of products. It concerns a molecular toolbox consisting of various analytical techniques, including anchor-pcr fingerprinting, which ultimately allows for a confirmation (detection) and identification of a UGM. Strategy 2 is based on the systematic gathering of knowledge with respect to GGO product composition and authorisation status, so that an efficient selection of potential unauthorised, suspect products can be made. This strategy employs the same technology as for strategy 1 (such as demonstrated for the UGM case study), but may lead to the optimisation of the monitoring through a targeted selection of products and analytical testing and shifts the use of analytical tools from screening to `blind samples` to a confirmation of suspect products. Both strategies were put to a test and the proof-of-concept was demonstrated on a real life UGM case study. All was set out in two peer review publications. 12,13 Further research and additional tools were necessary for the development of a broad strategy, which integrates the PCR matrix screening model and the anchor-pcr fingerprinting model with, for example, whole genome analysis technologies, for the detection of GGOs including UGMs. Figure 3: Depiction of anchor-pcr with use of a specific combination of restriction enzyme (NcoI, Mbo) and anchor primer (red fragment), which renders a unique pattern. The anchor primers are used in sets of 3 primers in a `nested` configuration (green, blue and black), each with another fluorescent label. The use of primers in a `nested` configuration is directed towards an increasing of the specificity and sensitivity of the anchor-pcr. Anchor-PCR is followed by fragment analysis through a separation of the products via capillary electrophoresis (CGE). The unique triplet of anchor-pcr amplicons allows for a unique identification of an event. 17

5 Figure 4: Genome walking via anchor-pcr. Amplification of sequences which flank screening elements leads to an identification of all present GGOs and constitutes the positive proof for the presence of a UGM. Figure 5: Depiction of 2 alternative approaches for the detection of UGMs. A standard analysis of GGOs is presently effectuated on the basis of known sequences, to obtain information with respect to the product composition by use of various analytical steps (above-left; below left triangle; left-hand arrow). This way of working is however becoming increasingly more difficult, taking into account the increasing number of GGOs under development, and the increasing complexity (which events to test, which not? Which tests/ analytes are to be applied, which not?). That s why there is an alternative procedure, starting from below, at product level (below right triangle; above-right; right-hand arrow). Product-based UGM discovery becomes possible on the basis of documented evidence of the unauthorised presence of GGOs. 18

6 References: 1. Van den Bulcke, M., et al Transgenic Plant Event Detection. PCT/EP2008/ [WO/2008/092866] Van den Bulcke M, et al. Anal Bioanal Chem Barbau-Piednoir E,et al. European Food Research and Technology 2010;230: Tse C, Capeau J. Ann Biol Clin (Paris) 2003;61: Community Reference Laboratory (CRL) Status of dossier web-page Waiblinger HU, et al. European Food Research and Technology 2008;226: Reiting R, et al. Journal fur Verbraucherschutz und Lebensmittelsicherheit 2007;2: Fernandez S, et al. J AOAC Int 2005;88: Höhne M, et al. European Food Research and Technology 2002;215: Corbisier P, et al. Anal Bioanal Chem 2005;383: Pardigol A, et al. European Food Research and Technology 2003;216: Ruttink T, et al. Anal Bioanal Chem 2010;396: Ruttink T, et al. Analytical and Bioanalytical Chemistry 2010;396: