Detection of genetically modified organisms

Abstract

This invention concerns a method for detecting genetically modified organism (GMOs) comprising transgenes and / or detecting genetically modified (GM) material in a sample comprising biological material, comprising performing a nucleic acid amplification reaction on nucleotide sequence elements extracted from an organism or sample with one or more first primer pairs, each of the one or more first primer pairs being specific for a polynucleotide sequence comprised in a transgene or comprised in a consensus sequence derived from an alignment of at least two homologous polynucleotide sequences of a transgene, to produce a first amplicon, and performing a nucleic acid amplification reaction with said extracted nucleotide sequence elements with one or more second primer pairs, each of the one or more second primer pairs being specific for a polynucleotide sequence comprised in a DNA barcode gene suitable for taxonomic identification. A first amplicon obtained from the reaction with the first primer pair is diagnostic of the presence a transgene, and the second amplicon obtained from the second primer pair allows for determination of the taxonomic identity of the organism or the origin of the material derived therefrom in a sample. The invention also concerns primer pairs and diagnostic kits for detecting the presence of GMOs.

Description

Detection of genetically modified organisms Technical domain

[0001] The present invention concerns the detection of the presence ofa transgene in a genetically modified organism (GMO) or in a samplecontaining genetically-modified (GM) material.Related art

[0002] The production of GM organisms and materials has been risingand becoming more widely available commercially, which has been prompting governments to implement strict legislation on the planting,marketing, labelling and trade of GM organisms and materials. Forensuring the introduced legislation, it is essential to develop accuratemethods for the detection of GM organisms or materials itself and to assurecompliance with threshold levels of GM products allow.

[0003] Any material deriving from GM crops may be identified bytesting for the presence of introduced DNA. Transgenic elements, ortransgenes, that are often found in commercialized GM crops usuallyconsists of several elements (promoters, terminators, genes, antibiotic resistance cassettes) that help screening for their presence.

[0004] There is a broad range of methods for genetically-modified (GM)DNA detection, and quantitative polymerase chain reaction (qPCR) approaches remain the most common. qPCRs allow the detection, identification, and quantification of genetic modifications, based on PCR amplification recorded in real-time. To further facilitate the detection of several DNA targets in a single reaction, multiplex PCR-based methods were developed. UniNE-8-PCT

[0005] Today detection of transgenes in GMOs relies primarily ontargeting a specific known sequence of a transgene and making a statement as to the presence of this targeted sequence in an organism or a biological sample.

[0006] However, establishing reference databases for transgenesremains extremely challenging as public disclosure of transgene sequencesis often incomplete.

[0007] In some jurisdictions like the European Union, there is anobligation to make available a detection method specific to the transgene, for example a GM event, when providing a GMO on the market. Detection methods for these transgenes rely therefore on the specific detection method provided for the GMO.

[0008] As the common practice usually is centred on the use of qPCR,often only primer sequences are made available, limiting the detection of GM events by only detecting the GM events when a certified set of primers is available. The dependency of a certified set of primers also restricts the completeness of the GM event sequences to be recovered, because there is no guarantee that the primers are recovering the complete GM event.

[0009] Previous studies using multiplex detection of different transgenestherefore relied on known amplification primers designed for methodsused in routine GMO feed samples screening strategies approved in GMOreference material (Scholtens, et al. 2017; Analytical and BioanalyticalChemistry 409 (15): 3877–89; https: / / doi.org / 10.1007 / s00216-017-0333-7) and( Arulandhu, et al.2018; Food Control 93 (November): 201–10; https: / / doi.org / 10.1016 / j.foodcont.2018.06.014).

[0010] In order to comply with regulatory and legal requirements, it ishowever highly desirable to identify the presence of any type of artificialtransgenic element in an organism or material comprising biological matter rather than selecting and targeting only specific transgenes. A moreUniNE-8-PCTcomprehensive and reliable approach for the detection of geneticmodifications to assess the presence of transgenes in GMOs or GM materialis therefore needed to better correspond to the legal and regulatoryrequirements. Short disclosure of the invention

[0011] It is an aim of the present invention to provide a method fordetecting GMOs and / or GM material that overcomes the shortcomings andlimitations of the state of the art.

[0012] It is another aim of this invention to improve traceability of GMmaterial and GMOs.

[0013] It is yet another aim of this invention to provide for analternative detection method of transgenic elements in biological material and / or organisms.

[0014] According to the invention, one or more of these aims areattained by the object of the attached claims, and especially by the independent claims. Optional embodiments of this invention are provided in the dependent claims.

[0015] In particular, one or more of these aims are attained by detectinga transgenic element in a genetically modified organism (GMO) and / or in asample comprising genetically-modified (GM) material, by providingnucleotide sequence elements extracted from an organism or from asample containing biological material and contacting said extractednucleotide sequence elements with one or more first primer pairs, each ofthe one or more first primer pairs being specific for a polynucleotidesequence comprised in a transgene or comprised in a consensus sequence.UniNE-8-PCT

[0016] The term “nucleotide sequence element” as used herein includesgenomic DNA as well as plasmids, chromosomal DNA, as well asextrachromosomal DNA.

[0017] A consensus sequence is a sequence which is derived from analignment of at least two homologous polynucleotide sequences of a transgene.

[0018] In addition, the extracted nucleotide sequence elements arecontacted with one or more second primer pairs, each of the one or moresecond primer pairs being specific for a polynucleotide sequence comprisedin a DNA barcode gene suitable for taxonomic identification. A specificbarcode sequence may for example be ubiquitous to a genus, a species or astrain.

[0019] The term “barcode sequence” refers to a standardized region ofthe genome, which may be used for to provide accurate species identification or identification of or taxonomic relationships on the basis of sequence variation within this sequence.

[0020] The first and the second primer pairs are amplification primerpairs.

[0021] Nucleic acid amplification reactions are performed on theextracted nucleotide sequence elements using at least one first and at least one second primer pair. Amplification using the first primer pair results inthe production of a first amplicon. Amplification using the second primerpair results in the production of a second amplicon.

[0022] The amplification reactions using the first primer pair and thesecond primer pair may be performed separately.UniNE-8-PCT

[0023] Provided the two primer pairs are suitable for multiplexing, theamplification reactions using the first primer pair and the second primer pair may also be performed in the same amplification reaction. It is also possible to use more than one first primer pair and / or more than one second primer pair in a multiplexed amplification reaction, provided the primer pairs are chosen to allow for multiplex amplification.

[0024] Based on the amplicons produced in these amplification reaction,a further comprising making a statement regarding the taxonomic identity, for example the species, of the organism or sample and about the presence of a transgene in said organism or sample can be made.

[0025] The first and / or the second primer pair may be chosen to produceamplicons of 100 to 500 nucleotides, or of 250 to 350 nucleotides. Amplicon of this length are particularly advantageous for detection and / or for sequencing.

[0026] In a subsequent step, the presence and / or the quantity of the firstamplicon and of the second amplicon is detected. The amplicons may be detected using detection methods commonly known in the art.

[0027] The presence of the first amplicon is diagnostic of the presence ofa transgenic element, or transgene, in the biological organism or in thesample containing biological material. The transgene may be a GM event ina plant or fungal species.

[0028] A GM event is defined by the insertion of DNA into a genome,for example a plant genome, as a result of a single transformation process.Multiple DNA sequences may be inserted during a single transformation process.UniNE-8-PCT

[0029] The second amplicon is diagnostic of the taxonomic identity, forexample the species, of the analysed organism, respectively of the genetic origin of the biological material contained in the analysed sample.

[0030] In one embodiment the quantity of the first amplicon isdetermined.

[0031] It is possible to compute the copy number of a specific transgeneby determining the amount of the first amplicon obtained using a primer pair specific for a sequence comprised in said transgene and comparing the amount of the first amplicon to the amount of second amplicon obtained in an amplification reaction having the same or substantially the same number of amplification cycles. Substantially the same in this context means no more than ± 10% of the amplification cycles performed with the first primer pair.

[0032] To determine the abundance of the amplified products in theform of reads the products may be sequenced. The term “reads” refers tothe sequences obtained from sequencing the amplicons, which representthe number of times a particular gene, e.g. a transgene or barcode gene, isobserved in the sequencing data. By comparing the number of reads of the transgene to the number of reads of the barcode gene, a ratio whichindicates the relative abundance of the transgene is obtained.

[0033] To give an example, if 10 reads are obtained for the transgeneand 10 reads for the barcode gene, the ratio is 1, i.e.10 / 10, indicating that the transgene is present in a similar proportion to the barcode gene. A ratio of 1 or more is diagnostic of the presence of a transgene in the organism.

[0034] However, if only 1 read for the transgene and 100 reads for thebarcode gene is obtained, the ratio is 0.01, i.e. 1 / 100, which suggests thatthe transgene is present in much lower amounts relative to the barcode gene. This ration could indicate that the single transgene read might be anUniNE-8-PCTerror or 'noise' rather than a true representation of the transgene's presence. A ratio this low is not directly indicative of a GMO.

[0035] The key aspect of this method standardization is not the absolutenumber of reads, but the ratio of transgene reads to barcode gene reads. This ratio helps us determine the presence and relative quantity of the transgene accurately, regardless of the total number of reads.

[0036] The copy number of portion of the barcode gene amplified usingthe second primer pair is known. In this approach, the second amplicon isused as an internal standard to quantify the number of transgenes in the sample.

[0037] Similarly, it is also possible to compute the number of transgenicelements present in the analysed organism or sample, if one or more first primer pairs are used which are either specific for a number of different transgenes, or which are designed using a consensus sequence derived from an alignment of at least two homologous polynucleotide sequences of a transgene.

[0038] The combination of reliable detection of transgenic elements inan organism of in a sample containing biological material allows for an improved traceability of the GM material, since it not only detects the presence of a specific or a variety of transgenes in biological material but also enables to identify the origin of the biological material on a taxonomic level. This is particularly useful if the identity of the biological material is unknown or not easily determined. By way of example, this method notonly permits for the detection of GM material in processed food or seeds,but it also genetically identifies the seed or identifies the genetic origin of the biological material contained in the processed food. In addition, depending on the specificity of the first primers chosen for the amplification reaction, the identity of the transgenic element(s) in said GM material can be determined.UniNE-8-PCT

[0039] In one embodiment at least one of the first primer pairs is specificfor a consensus sequence or for a sequence which is at least at least 95%, orat least 98% identical to a consensus sequence, and another first primerpair is specific for a sequence of a portion of a transgene which is nothomologous to this consensus sequence.

[0040] The first primer pair specific for the consensus sequence is used todetect a variety of homologous sequences in different transgenic elements. These homologous sequences are preferably sequences which are commonly used in the construction of artificial transgenic elements. Such sequences may for example be promoters.

[0041] The first primer pair specific for sequences of the transgenewhich are not homologous to a consensus sequence are generally more specific for a particular transgene. Such sequences may for example be found in a structural gene which encodes for the amino acid sequence of a polypeptide introduced into a GMO.

[0042] By combining a first primer pair specific for a consensus sequenceand another first primer pair specific for a sequence which is specific for a particular transgene, a statement can be made about the presence or the amount of transgenetic elements in the GM organism and material in general, as well as the presence or distribution of specific transgenetic elements in the GM sample. Thereby, a general assessment of the GM sample is combined with a more granular statement regarding theidentity and potentially the composition of different transgenes found in the sample.

[0043] The first primer pair may be directed to a specific locus in aconsensus sequence.

[0044] In one embodiment at least one first primer pair is specific for alocus having a nucleotide sequence selected from a group of sequencesconsisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ IDUniNE-8-PCTNO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10,SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15,SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20,SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25,SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30,SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35,SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40,SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45,SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50,SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55,SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60,SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65,SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70,SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75,SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80,SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85,SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90,SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95,SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100,SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ IDNO 110, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119,SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ IDNO 129, SEQ ID NO 130, SEQ ID NO 131, SEQ ID NO 132, SEQ ID NO 133, SEQID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138,SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141, SEQ ID NO 142, SEQ ID NO143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ IDNO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151, SEQ ID NO 152, SEQID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157,SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161, SEQ ID NO162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQ IDNO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171, SEQID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176,UniNE-8-PCTSEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO181, SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ IDNO 186, SEQ ID NO 187, SEQ ID NO 188, SEQ ID NO 189, SEQ ID NO 190, SEQID NO 191, SEQ ID NO 192, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195,SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 198, SEQ ID NO 199, SEQ ID NO200, SEQ ID NO 201, SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 204, SEQ IDNO 205, SEQ ID NO 206, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQID NO 210, SEQ ID NO 211, SEQ ID NO 212, SEQ ID NO 213, SEQ ID NO 214,SEQ ID NO 215, SEQ ID NO 216, SEQ ID NO 217, SEQ ID NO 218, SEQ ID NO219, SEQ ID NO 220, SEQ ID NO 221, SEQ ID NO 222, SEQ ID NO 223, SEQ IDNO 224, SEQ ID NO 225, SEQ ID NO 226, SEQ ID NO 227, SEQ ID NO 228, SEQID NO 229, SEQ ID NO 230.

[0045] It is however also possible to detect the presence of thetransgene by performing a hybridisation with a nucleotide sequence which is complementary to at least a portion of the targeted gene loci. The invention also concerns a nucleotide sequence complementary to any of the sequences SEQ ID NO 1 to SEQ ID NO 230 used for detection of a transgene.

[0046] The complementary nucleotide sequence and / or theamplification

[0047] This invention also concerns an amplification primer pair, which isspecific for a sequence having at least 95%, or at least 98%, or 100% sequence identity to any of these sequences SEQ ID NO 1 to SEQ ID NO 230.

[0048] In one embodiment the second primer pair is specific for aninternal transcribed spacer (ITS) sequence, for example ITS-1, or a portion thereof, or wherein the second primer pair is specific for a sequenceencoding the ribulose bisphosphate carboxylase L-subunit (rbcL) or aportion thereof. UniNE-8-PCT

[0049] The second primer pair may for example be one of the primerpairs disclosed in table 2 of Gholave et al (Gholave A.R. et al; 2017; PhysiolMol Biol Plants; 23(1): 155–167;doi: 10.1007 / s12298-016-0400-0) or by Kresset al (Kress, W. John.2017. , Journal of Systematics and Evolution 55 (4):291–307. https: / / doi.org / 10.1111 / jse.12254), which is specific for an ITS-1sequence. Barcode primers specific for ITS used in this study are provided asSEQ ID NO 246, SEQ ID NO 247, SEQ ID NO 248, SEQ ID NO 249, SEQ ID NO 250, SEQ ID NO 251, SEQ ID NO 252, SEQ ID NO 253, SEQ ID NO 254, SEQ ID NO 255, SEQ ID NO 256, and SEQ ID NO 257.

[0050] Alternatively or additionally, a second primer pair may be one ofthe primer pairs disclosed in table 2 of Gholave et al (Gholave A.R. et al;2017; Physiol Mol Biol Plants; 23(1): 155–167;doi 10.1007 / s12298-016-0400-0)or by Kress et al (Kress, W. John.2017. , Journal of Systematics and Evolution 55 (4): 291–307. https: / / doi.org / 10.1111 / jse.12254), which isspecific for an rbcL sequence. Barcode primers specific for rbdL used in thisstudy are provided as SEQ ID NO 231, SEQ ID NO 232, SEQ ID NO 233, SEQ ID NO 234, SEQ ID NO 235, SEQ ID NO 236, SEQ ID NO 237, SEQ ID NO 238,SEQ ID NO 239, SEQ ID NO 240, SEQ ID NO 241, SEQ ID NO 242, SEQ ID NO243, SEQ ID NO 244, and SEQ ID NO 245. .

[0051] In one embodiment, the first and / or the second amplicon issequenced. The amplicons may for example be sequenced using next generation sequencing (NGS).

[0052] Determining the sequence of the first amplicon allows fordetermination of the genetic origin of the transgene by comparing the sequence of the amplicon to known sequences of transgenes and / or biological organisms.

[0053] Determining the sequence of the first amplicon also allows fordetection of discrepancies between the sequence of the amplicon and the target sequence in the transgene. In this way, further genetic modifications UniNE-8-PCTin the transgene, for example point mutations, single nucleotide polymorphisms (SNPs), and / or gene editing events, can be detected.

[0054] The organism tested for the presence of transgenic elements mayfor example be a plant, an animal, a protozoa, or a fungi. It may also be a prokaryotic organism, such as a bacterium.

[0055] The sample containing biological material is not particularlylimited. It may for example be foodstuff, for example processed food, a beverage, feed for animals, for example feed for livestock, a fabric or processed material for the production the fabric.

[0056] The invention also concerns a diagnostic kit for detecting thepresence of a transgene in an organism or in a sample comprisingbiological material, comprising one or more first primer pairs, each of theone or more first primer pairs being specific for a polynucleotide sequencecomprised in a transgene or comprised in a consensus sequence derived from an alignment of at least two homologous polynucleotide sequences of a transgene, and one or more second primer pairs, being specific for apolynucleotide sequence comprised in a DNA barcode gene suitable fortaxonomic identification.

[0057] One or more of the first primer pairs may be specific for asequence comprised in the group of sequences consisting of SEQ ID NO 1 to SEQ ID NO 230. Short description of the drawings

[0058] Exemplar embodiments of the invention are disclosed in thedescription and illustrated by the drawings in which: UniNE-8-PCTFigure 1A illustrates schematically an exemplary structure of a GMevent, typically comprising a promotor (A), junctions (B), protein-coding region (C) and a terminator (D);Figure 1B illustrates schematically selected loci (shown as black boxes)in the aligned sequences and primer pairs shown as arrows used forproducing amplicons; Figure 2A is an overview of the length of the 83 designed GM amplicons; Figure 2B is a representation of the number of samples per plant species, giving a total of 192 samples that include 92 different individuals GMO and controls, dilutions and replicates;Figures 3Ato 3J show assessments of known GMO events by ampliconsequencing and contrast to control samples, wherein the following GM events were covered for multiple segments (with base pair positions indicated for multiple amplicons covering the samesegment of an event): Events (Figure 3A) MON89034, (Figure 3B)BT11, (Figure 3C) DAS59122, (Figure 3D) MON88017, (Figure 3E) MON88017, (Figure 3F) MON87427, (Figure 3G) MON810, (Figure 3H) SYN3272, (Figure 3I) MON87460 and (Figure 3J) MON15985.Figures 4A to 4E show results of an assessment of differentconcentrations of GM and dilutions of sample DNA, whereinFigure 4A shows a GM event MON810 spanning three assessedregions (3’, 5’ and hsp70),Figure 4B shows a GM event called SYN3272, composed of threerecovered regions (amy797E, PEPC9-intron, and T35S-CaMV) in different locus positions where the reads were aligned and we UniNE-8-PCTnormalized the count in bp, for the 0.98% concentration of GMO in white, the 9.8% concentration of GMO maize in black and the non- GMO maize (crossed lines), Figure 4C shows a GM event called DAS59122 amplified at four different loci.Figure 4D shows a normalized read count for a GM event calledMON88017 in different DNA dilutions, sample no diluted, diluted 10x, diluted 100x and diluted 1000x;Figure 4E shows a normalized read count for a GM event calledMON88017 in different DNA dilutions, sample no diluted, diluted 10x, diluted 100x and diluted 1000x;Figure 4F shows a linear regression of the concentration of the DNAin dilutions versus the normalized read counts of the GM event called MON88017;Figures 5A, 5B, 5C, 5D, 5E, 5F and 5G show amplifications of GMevents included in the databases euginius, portugene and Fraiturewith: Heatmap for 11 samples including nine different GMO eventsfor maize with one non-GMO maize as control, one GMO event forcotton with one non-GMO cotton as control, aligned to the 83 different designed amplicons from the three databases (euginius,portugene and Fraiture, et al. 2019; Scientific Reports 9 (1): 7141.https: / / doi.org / 10.1038 / s41598-019-43463-5) and read counts normalized using barcoding loci sequencing depth. UniNE-8-PCTExamples of embodiments of the present invention Resultsfrom the ampliconIn order to test the feasibility and assess the performances of the amplicon- sequencing assay to detect transgenic material in food, feed or seed matrices, we gathered a total of 115 consensus GM sequences from various sources: EUginius (EUropean GMO INItiative for a Unified Database System, (“The European GMO Database” n.d.)) and portugene (Moreira, Carneiro,and Pereira 2017a) databases. In addition, we manually added threesequences from a gene encoding the dihydroflavonol 4-reductase from an unauthorized GM Petunia recently detected in Fraiture, et al.2019; Scientific Reports 9 (1): 7141. https: / / doi.org / 10.1038 / s41598-019-43463-5. For the design of amplicons, we retrieved the GM consensus sequences if multiple redundant sequences were found in databases. Figure 1A schematically depicts an exemplary structure of a transgenic element, inthis case a GM event, comprising a promotor (A), junctions (B), protein-coding region (C) and a terminator (D).

[0059] Consensus sequences which were longer than 300 bp werefragmented to design multiple similarly spaced loci to produce amplicons . Figure 1B schematically depicts three aligned sequences, indicated by vertical lines, and selected loci used for amplification shown as black boxes. Primer pairs are indicated as black arrows, the presentation is not to scale.

[0060] Based on this GM sequence set, 230 primer pairs for GMsequences that correspond to 83 unique GM sequences were designed and 15 rbcL + 12 ITS primers (previously published by Kress, W. John.2017. , Journal of Systematics and Evolution 55 (4): 291–307; https: / / doi.org / 10.1111 / jse.12254) were added for plant species identification. Following the manufacturer's instructions, we aimed for an amplicon length of ~200bp and we obtained a maximum length of 240 bp UniNE-8-PCTfor 14 GM locus and the smallest amplicons with a length in the range of 69-199 for 21 GM loci.

[0061] The amplicon-sequencing assay was performed using amicrofluidics platform and aimed generally at detecting, identifying and potentially quantifying genetically modified events. In a single flow cell run, a total of 186 samples including replicates, representing 92 distinct samples of GMO and non-GMO control material, were analysed. The speciescovered were alfalfa (n=10) (Medicago sativa), beetroot (n=6) (Betavulgaris), canola (n=23) (Brassica napus), cotton (n=26) (Gossypium sp),creeping (n=3) (Beta vulgaris), linseed (n=3) (Linum usitatissimum), maize(n=66) (Zea mays), potato (n=6) (Solanum tuberosum), rice (n=3) (Oryzasativa), soybean (n=37) (Glycine max) and wheat (n=3) (Triticum),additionally blank (n=6) (Figure 2B). Next generation sequencing using Illumina NextSeq 550 generated a total of 80,478,507 reads after trimming and merging overlapping read pairs per sample and locus.

[0062] For every sample, the reads were aligned to the 115 targetsequences and plant-barcoding sequences rbcL and ITS sequences of the 11species. One sample of maize obtained a maximum of 3,122,899 alignedreads. The minimum number was 229 aligned reads which correspond to asample coming from soybean. The other three minimum aligned reads ranged from 663 to 1418 and correspond to 3 replicates of DNA dilution (1000x) of a maize sample (Figure 2B). Identification ofamplification ofloci

[0063] An important checkpoint for the identification of plant GMOs isto determine the species' identity. We incorporated in our targetingsequencing assay two sets of primers ( Kress, W. John. 2017, Journal ofSystematics and Evolution 55 (4): 291–307. https: / / doi.org / 10.1111 / jse.12254)previously recognized as plant DNA barcodes that amplify the rbcL and ITSloci. Given that the 15 rbcL + 12 ITS primers used in this study areamplifying in different regions of rbcL and ITS, it was decided to work withUniNE-8-PCTthe maximum number of mapped reads per barcode, which were correlated to the mean (r = 0.897***, Pearson correlation coefficient), median (r = 0.848***) and mode (r = 0.848***). To compare the success ofrbcL and ITS amplification the max number of mapped reads for the 186samples was taken. RbcL max count was significantly positively correlatedto ITS max count for alfalfa, canola, cotton, potato and soybean, being in the range of 0.983*** (*** meaning p-value <0.001) to 0.748*** (Table 1).In the cases of beetroot, creeping, rice and wheat, the correlation was notsignificant (p-values > 0.05), given that a low number of samples, between 3 and 6 samples was provided. The case of maize in particular, because there is not a positive correlation: 0.503***. (Table 1). We also compared the max barcode count against the total aligned counts to assure that the general quality of the samples has been captured by the plant barcodes. For ITS, 10 plant species showed a positive significant correlation from 0.88*** to 1***, with the only exception of beetroot with a no correlationof 0.25**. RbcL varies more between species with a range of -0.96 p=0.19 to0.97***. CorrelationBy species ITS vs rbcLAlfalfa 0.874***Beetroot -0.294Canola 0.863***Cotton 0.846***Creeping 0.425UniNE-8-PCTLinseed -0.997Maize 0.503***Potato 0.983***Rice 0.316Soybean 0.748***Wheat 0.838Table 1: Correlations of the maximum read count for ITS compared to rbcLfor all soybean samples including a table of correlations for other plant species.

[0064] Every sample was mapped to the 11 plant species sequences ofrbcL and ITS. We assigned species identities according to the highest readdepth among rbcL and ITS reference sequences. As the species was knownfor all samples, we assessed the power of the assay to recover the true species. Only using, the ITS primers we achieved 91 out of 186 correct predictions of the species identity corresponding to an accuracy of 48.9%.For the rbcL primers, we achieved 182 out of 186 correct predictions of thespecies identity corresponding to an accuracy of 97.8% . Hence, rbcL primers predicted more accurately the species and we used this barcoding loci for most species. The species that performed best was maize showing the highest correctly mapped reads ratio for 13 samples (79-172), meaningthat the primers used to amplify rbcL and the rbcL sequence used to alignare providing the best correct mapping. The worst correctly mapped readsratios correspond to the three rice samples: 0.912- 0.686.Detection of GM events based on amplicon sequencing assay UniNE-8-PCT

[0065] 98 individual samples including 11 non-GMO controls wereanalyzed. The samples were known to carry at least 10 sequence fragments from a specific GM event with 9 samples originating from maize and one from cotton.10 GMO samples were analyzed and compared against the non-GMO controls. To account for variation in total reads among samples and the uneven presence of GM sequences, read counts were normalized using normalized mean depth by the maximum read depth at thebarcoding locus. In the case of maize, rbcL was used because the readdepth was higher in comparison to ITS. For cotton, ITS was used because the read depth was higher for ITS. From the 10 analyzed GM events, six events (MON89034, BT11, DAS59122, MIR162, MON88017 and MON87427) amplified well in the GMO sample and showed no meaningful amplification in the non-GMO control (Figure 3A-F). For example, MON 89034 amplified well for 3’MON89034 (normalized count 0.094), 5’MON89034 (normalized count 0.004), hsp70-locus56 (normalized count 0.747), hsp70-locus307 (normalized count 0.285), LTa.lhcbl (normalizedcount 0.002), tahsp17 (normalized count 0.331) and CTP2 (normalized count0.405). For the three other GM events (MON810, SYN3272 and MON87460), the GMO maize also amplified more GM sequences compared to the non- GMO control (Figures 3G-I). For example, the sample MON810 where the non-GMO control is amplified in 5’ MON810-locus 59 (normalized count 0.383) vs the GM sample (normalized count: 0.526) (Figure 3G).

[0066] In order to investigate why some amplicons were amplified innon-GMO samples matching sequences to the amplicon on NCBI using BLAST were analysed. A close hit in a non-GMO maize genome, with theaccession number AC225944.3 having 91.89% of identity in a query of 222of length, was recovered. The last sample corresponds to MON15985 where the non-GMO produced 90.9741 normalized read counts surpassing the GMO cotton showing 48.72025 normalized read counts for the “chloroplast” labelled GM sequence amplicon of only 83bp (Figure 3J). A blast BLAST search on NCBI retrieved both chloroplast and nuclear genome matches, but not in cotton samples. In contrast, the MON15985 sequence labelled “Cotton_MON15985” amplified more in the GMO cotton (42.6551) compared to non-GMO cotton (10.184160 normalized counts; Figure 3J). UniNE-8-PCTThe most discriminant amplicon for detecting the MON15985 event was the Oriv amplicon.of the detection of the amplicon

[0067] For monitoring purposes, mixed GMO samples need to bescreened. To assess the power of the targeted sequencing assay of thisstudy, three GMO samples diluted in control DNA at various ratios weretested. For the GMO SYN3272 at the locus amy797E, the GMO (constituting9.8%) amplified in the range of 0.01- 0.002 normalized read countscontrasted with no recovered reads for the GMO mixed in at 0.98% (Figure4B). The sample DAS59122 amplified in both the 1% mixture in the rangeof 1.15 e-10 – 0.0021 and the 10% mixture in the range of 0.00018 –0.00796 (Figure 4C). Unexpectedly, MON810 showed more amplification forGMO 1% compared to GMO 10% (Figure 4A). For example, 5’ MON810locus 336 amplified for GMO 10% 1.2830 compared to 1.553 normalized read counts amplified for GMO 1%. The non-GMO control sample showed however also 0.7572 normalized read counts.

[0068] Next, the sensitivity of the amplicon assay to detect DNA diluted10x, 100x, and 1000x was assessed. Sample MON88017 was tested, however,the normalized counts mismatched the expected trend from the dilutions. The most likely explanation for this variability is the overall low number of reads likely producing noise (Figure 4D, Figure 4E). Using the mean depth of mapped reads (before normalization) showed an expected change in read depth following the dilution series (Figure 4F). Additional amplicons for GM event

[0069] 73 target sequences were recovered from EUgenius, portugeneand Fraiture, et al. 2019; Scientific Reports 9 (1): 7141.The sequences were amplifiedin the amplicon-sequencing assay of this study. The amplification of the 10UniNE-8-PCTGM sequences described above was measured. The log normalized countsof these gene events are shown in Figures 5A to 5G.

[0070] MON15985 cotton is amplifying the sequence Cotton_MON15985as expected and also OriV (Figure 3J). Non-GMO controls showed amplification of multiple 3' and 5' GM flanking sequences as expected from the above findings. Three additional sequences including “FB707511.1 cry1A.105”, “DL476427.1 CORN EVENT” and “aadA” showed amplification in the MON15985 GMO. In regards to the GMO maize samples, MON 89034 was amplifying the sequences 3’MON89034, hsp70, tahsp and CTP2 (Figure 3A). Beyond amplified flanking sequences, three sequences amplified in the GMO including “FB707511.1 cry1A.105” and “DL476427.1 CORN EVENT”, and “dihydroflavonol4-reductase_MF521566.1”, which the MON15985 GMO was not known to contain. Discussion

[0071] qPCR has been the historical “gold standard” of GMO detectionin all matrices that could be circulating: food, feed, seeds and in the environment. High sensitivity but expensive and labour-intensive, not fit to the ever-increasing list of GM commercialized. Improving methods for detecting GMOs is therefore important (for the implementation ofgovernmental regulations, trade, and biosafety). In this study the feasibilityof using a microfluidics-based tool for the detection of GM events wasinvestigated. A set of 230 amplicons was selected, whichth represent 83unique GM events. In addition, 27 plant barcoding primers were allowed toprecisely identify species contained in the samples. The advantage of using amplicon sequencing is the extreme versatility of its design (can be customized repeatedly) and taking advantage of the broad range of amplicons, it allows upfront monitoring of samples without prior knowledge of the genetic modification.

[0072] 27 primers were used to amplify barcoding genes rbcL and ITSthat wre used to identify the plant species contained in the sample. TheUniNE-8-PCTsamples corresponding to cotton were however not performing well: onlyrelatively low read counts were mapping to the rbcL gene. Low coveragemay be due to the selected primers to amplify the loci performed poorly on those samples. The complexity of amplifying a barcoding locus using multiple overlapping pairs of primers was apparent in the read mappingpatterns along the rbcL and ITS sequences. Replacing the pool of barcodingloci primers with pairs of custom-designed primers for a specific range of species would alleviate the complexity in amplification and likely increase consistency in amplification across the desired species.

[0073] The detection of GM events in positive control samples wassuccessful for a wide range of GM sequences. However, non-GMO controls amplified in some GM regions including 3’ and 5’ flanking sequences, the promoters, and terminators, which likely have homology to regions in the genome outside of the GM sequences. Such a lack of specificity in flanking and promoter sequences of GM events against regular plant DNA sequences can explain why GMO event promoters are also amplifying in non-GMO plants. The GM regions, which were amplified in non-GMO samples showed indeed sequence homology in genomes of non-GMO plants. This supports the lack of specificity in the 3’ and 5’ flanking sequences of GM events. However, the recovered homologous sequences in plant genomes were not showing 100% identity, which means the SNP calling could be used to differentiate between reads mapping to a GMevent versus reads mapping to non-GMO sequences elsewhere in thegenome.

[0074] The lack of an entire amplicon sequence and material accessibilityis challenging for producing specific sets of primers covering GMO sequences comprehensively (Moreira, Carneiro, and Pereira 2017a). Our study shows that a de novo design of GM amplicons is feasible using public sequence information and that a broad range of potential GM sequences can be assessed in parallel. The approach of a microfluidics-based targeted amplicon sequencing assay enables to screen both hundreds of samples (or replicates) simultaneously but also allows for large sets of primers to be included in parallel. In principle, the microfluidics chips would allow the UniNE-8-PCTpooling of thousands of primer pairs for single-step amplifications. Given the uncertainty of amplifying specific sequences from unknown samples and modification events, the sequence information provides significantly greater certainty about the identity of an amplified sequence. This is in contrast to qPCR approaches that lack direct validation capabilities under non-standard conditions.

[0075] In contrast, targeted amplicon sequencing is less sensitivecompared to qPCR. In our analyses, we found reliable amplification to ~1:100 dilutions, at lower concentrations the detection is likely to become poorly reproducible. Such detection limitations can be remedied partially by increasing the overall sequencing coverage of the amplicons as sensitivity is at least partially correlated with sequencing depth. Our work fits into recent efforts to standardize and propose a statistical frameworkfor the detection of GMOs (Willems et al. 2016) based on the number ofreads aligned per sample. Our presented workflow expands the capabilities by targeting a large number of sequences of interest specifically and allowing for the efficient detection of plant species present in a sample.

[0076] In conclusion, our microfluidics-based targeted amplicon assayallows the simultaneous monitoring of multiple genetic modifications and demonstrates how next-generation sequencing techniques and data analysis can improve the capacity of environmental surveys and food supply screening. Our study shows the relevance of amplicons sequencing that can be realistically implemented into GMO detection and efficiently analyzed using a structured bioinformatics pipeline. Materials and Methods Collection of samples

[0077] Genotyping was performed on 92 plant samples, mostlyconsisting of GMO certified reference materials. The samples covered eleven of the most widely cultivated plant species worldwide, including UniNE-8-PCTmaize (Zea mays), soybean (Glycine max), canola (Brassica napus), cotton (Gossypium sp), alfalfa (Medicago sativa), potato(Solanum tuberosum), beetroot (Beta vulgaris), creeping bentroot (Agrotis stolonifera), linseed (Linum usitatissimum), wheat (Triticum) and rice (Oryza sativa). The samples also included non-GMO plants used as negative controls. To assess the sensitivity of the assay, different concentrations of the same GM plants were used, typically ranging from 0.98 % to 100 % (ratio of the GM plant species in the total plant species, expressed in mass / mass, as equivalent of copies / copies of haploid plant genomes). DNA extraction

[0078] DNA extractions were carried out using the NucleoSpin Plant II kit(Macherey-Nagel,GmbH, Germany) following the manufacturer's protocol. DNA concentrations of all samples were assessed using a NanoDrop One spectrophotometer (Thermo Scientific). The DNA concentration of all thesamples were standardized by diluting them in water to 50 ng µl­1. Toexplore the effects of low DNA input, one of the GMO maize samples wasdiluted in a dilution series starting from 50 ng µl­1 down to 5 ng µl­1 (10-fold), 0.5 ng µl­1 (100-fold) and 0.05 ng µl­1(1000-fold). For downstreamapplications, two additional replicates for 41 of the samples wereperformed, including the replicates for the dilution series.

[0079] GM sequences, which were likely to be GM sequences, wererecovered from two GMO databases, EUginius (EUropean GMO INItiativefor a Unified Database System) and portugene (Moreira, Carneiro, andPereira 2017a), and used as target sequences. In addition, sequencesencoding the dihydroflavonol 4-reductase gene from an unauthorized GMPetunia sequence ( Fraiture, et al. 2019; Scientific Reports 9 (1): 7141.were aded. To avoidredundancy between the target sequences, nearly identical sequences were clustered and multiple alignments using Clustal Omega-v1.2.3 (“Clustal UniNE-8-PCTOmega < Multiple Sequence Alignment < EMBL-EBI” n.d.) were produced.Aligned sequences were used to produce a consensus, retaining ambiguous bases and yielding a total of 115 unique sequences targeting specific GM events. To identify the plant species of tested samples, two previously published sets of primers (Kress, W. John.2017. , Journal of Systematics and Evolution 55 (4): 291–307. https: / / doi.org / 10.1111 / jse.12254) widely used asplant DNA barcoding loci for taxonomic identification were added. Theseprimers are specific for RbcL (the chloroplast-encoded large subunit of theRibulose-1,5-bisphosphate carboxylase-oxygenase) and ITS (InternalTranscribed Spacer), from the ribosomal DNA. Amplicon design

[0080] Of the 115 unique target sequences, longer sequences exceeding300 base pairs were segmented into multiple regions for individualamplicon design, for better optimization during amplification. After the segmentation 230 candidate loci (SEQ ID NO 1 to SEQ ID NO 230) for assayprimer design were obtained according to the Fluidigm Inc. procedures. The targeted amplicon length ranged from 62-240 bp reflecting constraints in conserved sequences and base composition (see Figure 2A). We obtaineda total of 230 pairs of primers corresponding to the GM target sequences. In the case of the barcodes that amplify rbcL and ITS genes, we compiled aset of 27 primers representing various amplicon designs covering the same loci corresponding to rbcL (SEQ ID NO 231 to SEQ ID NO 245) and ITS (SEQID NO 246 to SEQ ID NO 257).

[0081] Libraries were prepared following the manufacturer's protocolPN 101-0414 G1 for the Juno LP 192.24 integrated fluidic circuits plate (IFC;Fluidigm Corporation, San Francisco, CA, United States). After loading all reagents on the IFC, target amplicons were generated for each sample through PCR amplification on a specialized thermocycler (Juno system; Fluidigm). A total of 257 primers subdivided into 10X assay pools were UniNE-8-PCTcombined in the IFC with an inlet containing 2 µl of sample pre-mix, 2 µl gDNA (50ng / µl) and 1 µl Barcode primer (unique barcode per sample) consisting of a DNA sample and an individual barcode. After amplification, samples were pooled into a single tube and purified; the first clean-up is double-sided (0.4X / 0.9X Double-Sided SPRI), meaning it first (0.4X) removes fragments that are bigger than our targets, then (0.9X) it binds and selects our target by leaving back and washing off the smaller fragments. The second and third clean-ups are "normal" to remove excess primers (0.8XSPRI); to ensure the removal of excess primers before adapter ligation.Finally, sequencing adapters were added by PCR to the purified library followed by a final round of purification according to the manufacturer's protocol. The quantity and quality of the library were assessed using a Qubit fluorometer assay (ThermoFisher) and a 4200 TapeStation electrophoresis instrument (Agilent). The final library was sequenced on a single lane of a NextSeq 500 system (Illumina) in mid-output mode adding ~30% PhiX to reduce issues due to low sequence complexity.

[0082] An rbcL sequence of maize from NCBI (NC_001666.2) and an ITSsequence from potato (CP046695.1) were obtained. Matching rbcL and ITSsequences were then retrieved using BLAST (“Nucleotide BLAST: SearchNucleotide Databases Using a Nucleotide Query” n.d.) to complete a libraryof barcoding sequences for all eleven included plant species. The plant barcode sequences were added to the GM reference sequences. Raw read data were demultiplexed using bcl2fastq v-2.19.0.316 and trimmomatic v-0.36 (Bolger, Lohse, and Usadel 2014) was used for quality trimming.Forward and reverse reads were merged using flash v-1.2.11 (Magoč andSalzberg 2011). Merged reads were aligned to the reference sequenceusing Bowtie2 v-2.3.5 using the following settings: --very-sensitive-local -- phred33 (Langmead et al.2019). From the aligned reads, the depth per position and cut the positions that were not designed in the ampliconsusing samtools v-1.19 was calculates. Additionally, to verify that the chosenreference sequences used for the barcodes are correct all the nucleotide UniNE-8-PCTsequences for rbcL and ITS found on NCBI were downloaded, for each ofthe eleven plant species generating eleven fasta files for rbcL and elevenfasta files for ITS. The fasta files were used as a reference to align the merged reads by Bowtie2 v-2.3.5 using the following settings: --very- sensitive-local --phred33 (Langmead et al.2019). From the aligned reads, the depth per position using samtools v-1.19 was calculated and the depthto the previous depth of rbcL sequence from maize (NC_001666.2) and ITSsequence from potato (CP046695.1) was compared with the retrieved BLAST sequences of the eleven plants. NC_001666.2, CP046695.1 and their corresponding BLASTs were chosen given a higher depth value. UniNE-8-PCT

Claims

Claims1. Method for detecting genetically modified organism (GMOs) comprisingtransgenes and / or detecting genetically-modified (GM) material in a samplecomprising biological material, comprising -providing nucleotide sequence elements extracted from an organismor from a sample containing biological material,- contacting said extracted nucleotide sequence elements with one ormore first primer pairs, each of the one or more first primer pairsbeing specific for a polynucleotide sequence comprised in atransgene or comprised in a consensus sequence derived from analignment of at least two homologous polynucleotide sequences of a transgene, and performing a nucleic acid amplification reaction to produce a first amplicon, -detecting the presence and / or quantity of said first amplicon,wherein the presence and / or the quantity of said first amplicon is diagnostic of the presence of a transgene in the biological organism or in the sample comprising biological material, -contacting said extracted nucleotide sequence elements with one ormore second primer pairs, each of the one or more second primerpairs being specific for a polynucleotide sequence comprised in aDNA barcode gene suitable for taxonomic identification, andperforming a nucleic acid amplification reaction to produce a secondamplicon, -detecting the presence of said second amplicon and determining thetaxonomic identity, for example the species, of the organism, ordetermining the taxonomic identity of the organism or the origin ofUniNE-8-PCTthe material derived therefrom in a sample on the basis of saidamplicon.

2. The method of claim 1, further comprising making a statementregarding the taxonomic identity, for example the species, of the organism or sample and about the presence of a transgene in said organism or sample.

3. The method of claim 1 or claim 2, wherein at least one of the firstprimer pairs is specific for the consensus sequence or for a sequence whichis at least at least 95%, or at least 98% identical to the consensus sequence,and wherein another first primer pair is specific for a sequence of a portionof a transgene which is not homologous to the consensus sequence.

4. The method of any of claims 1 to 3, wherein at least one of saidconsensus sequence is at least 95%, or at least 98%, or 100% identical to apolynucleotide sequence comprised in a group of sequences consisting ofSEQ ID NO 1 to SEQ ID NO 230.

5. The method of any of claims 1 to 4, wherein one or more of the firstprimer pairs and / or one or more of the second primer pairs are chosen toallow for multiplex amplification.

6. The method of any of claims 1 to 5, wherein the second primer pair isspecific for an internal transcribed spacer (ITS) sequence, for example ITS-1,or a portion thereof, or wherein the second primer pair is specific for asequence encoding the ribulose bisphosphate carboxylase L-subunit (rbcL)or a portion thereof.

7. The method of any of claims 1 to 6, wherein the number of copies of adetected transgene in a genetically-modified organism (GMO) isdetermined by determining the ratio of the amount of the secondamplicon, which is used as an internal standard, and the amount of the firstUniNE-8-PCTamplicon produced with the same or substantially the same amount of amplification cycles.

8. The method of any of claims 1 to 7, wherein the first and / or the secondamplicon is sequenced.

9. The method of any of claims 1 to 8, wherein the first amplicon issequenced and wherein the genetic origin of the transgene is identified on the basis of said sequence. 10.The method of any of claims 1 to 9, wherein the first amplicon issequenced and wherein said sequence is compared to known nucleotidesequences of transgenes to detect the presence of mutations in thetransgene, such as point mutations, SNPs or gene editing events. 11.The method of any of claims 1 to 10, wherein the organism is a plant.12.The method of any of claims 1 to 11, wherein the sample is foodstuff,for example processed food, a beverage, feed for animals, for example feed for livestock, a fabric or processed material for the production thereof.13.An amplification primer pair specific for, or a nucleotide sequencecomplementary to at least a portion of a sequence having at least 95%, orat least 98%, or 100% sequence identity to any one of the sequences SEQID NO 1 to SEQ ID NO 230. 14.A diagnostic kit for detecting the presence of a transgene in anorganism or in a sample comprising biological material, comprising- one or more first primer pairs, each of the one or more first primerpairs being specific for a polynucleotide sequence comprised in atransgene or comprised in a consensus sequence derived from an UniNE-8-PCTalignment of at least two homologous polynucleotide sequences of a transgene, and -one or more second primer pairs, being specific for a polynucleotidesequence comprised in a DNA barcode gene suitable for taxonomic identification.15.The diagnostic kit of claim 14, wherein at least one of the one or morefirst primer pair is an amplification primer pair specific for a sequencehaving at least 95%, or at least 98%, or 100% sequence identity to apolynucleotide sequence comprised in a group of sequences consisting of SEQ ID NO 1 to SEQ ID NO 230. UniNE-8-PCT

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