Tel: +47 64970100; Fax: +47 64970333; Email: [email protected] ... sample and the amount of template DNA. ... of the different templates are maintained.
Nucleic Acids Research, 2003, Vol. 31, No. 11 e62 DOI: 10.1093/nar/gng061
A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quanti®cation of transgenic maize in food and feed Knut Rudi*, Ida Rud and Askild Holck MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 AAS, Norway Received January 31, 2003; Revised and Accepted April 9, 2003
ABSTRACT We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quanti®cation of multiple gene targets is desired. We used quanti®cation of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the ®rst few cycles bipartite primers containing a universal 5¢ `HEAD' region and a 3¢ region speci®c to each genetically modi®ed (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-speci®c exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligonucleotides hybridising to internal segments of the PCR products are then sequence speci®cally labelled in a cyclic linear signal ampli®cation reaction. This is done both to increase the sensitivity and the speci®city of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1±2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5¢ nuclease PCRs. There were no signi®cant differences in the quanti®cations using the two approaches. The samples could, by both methods, be quanti®ed as containing >2%, between 1 and 2%, between 0.1 and 1%, or 1% of any ingredient originate from GM material. Considering the large numbers of GM constructs expected in the future, multiplex quantitative measurements are required to determine whether the foods contain approved or unapproved GM ingredients, and whether the amount of GM material is above or below the 1% limit. GM constructs are often composed of common elements such as the P35 promoter, nos terminator and an antibioticresistance gene as selection markers (12). Detection of these elements indicates that GM material may be present. Construct-speci®c PCRs spanning a junction speci®c for a GM construct give more accurate determinations. Sometimes the same genetic elements are used in several different GM constructs of which only some may be approved for use in the EC. The optimal solution is therefore to design PCRs in regions that are event speci®c in order to obtain complete selectivity for a given construct. These regions may include the overlap between endogenous and inserted DNA or speci®c rearrangements arising unintentionally during the transformation event. We have developed a new quantitative multiplex DNA array based PCR method (MQDA-PCR). The method was used to quantify different transgenic maize in food and feed samples. The method is based on a two-step PCR (schematically shown in Fig. 1). In the ®rst step bipartite primers containing a universal 5¢ `HEAD' region and a 3¢ region speci®c to each construct were used. The PCR is run for a few cycles only. The unused primers are then removed with a single-strand DNA-speci®c exonuclease. This digestion removes the bipartite primers that would otherwise prime unwanted ampli®cations. The second step of the PCR is run containing only the primer consisting of the universal HEAD region. This step is thus competitive, conserving the ratio between the amplifying fragments. Finally, both to obtain better speci®city and sensitivity, a linear ampli®cation by sequence-speci®c labelling of DNA probes is included. The detection is done in a DNA array format through hybridising the labelled probes to their complementary sequences spotted on a solid phase (13,14). In our multiplex set-up, both common elements, construct-speci®c elements and elements that are speci®c for the different events were included (Fig. 2). The system also has a synthetic DNA as an internal reference standard for assessing both PCR inhibitory compounds in the sample and the amount of template DNA. MATERIALS AND METHODS Test materials Mon810, Bt11 and Bt176 certi®ed maize ¯our reference material prepared by the EU Joint Research Center, IRMM (Institute for Reference Materials and Measurements, Italy) were obtained from Fluka, Buchs, Switzerland. Additional Mon810 material was a gift from C. Bulkmans. For T25, GA21, CBH351 and DBT418 commercial maize reference material is not available. Larger amplicons encompassing the amplicon regions used in the multiplex assay were therefore mixed in various amounts with a background of non-GM maize DNA to create appropriate reference DNA material for these GM maizes. Food (corn meal, corn meal mixes, pop
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Figure 1. Schematic representation of the MQDA-PCR method. (1) In the ®rst PCR step, the targets are ampli®ed with bipartite primers containing universal HEAD regions (grey) which are equal for all the different targets. (2) The HEAD-containing primers are then removed by enzymatic digestion. (3) In the second PCR step, a primer identical to the HEAD sequence is used, creating a competitive reaction where the relative amounts of the different templates are maintained.
corn, corn snacks, canned sweet corn, whole kernel corn) and feed (horse feed, chicken feed, pet food) samples were from various food and feed stores in California, USA. DNA puri®cation Samples were homogenised with a warring blender 708A (Krups, Germany) and puri®ed using DNA adsorption columns (Dneasy plant mini kit; Qiagen, Hilden, Germany) as described by the manufacturer with the following modi®cations. The initial buffer volume was doubled and lysis was carried out for 30 min at 65°C using a shaking incubator. When eluting DNA bound to the column, 50 ml of elution buffer preheated to 70°C was used. In the repeated elution step another 50 ml buffer was added and the columns were spun at 13 000 r.p.m. (Biofuge Fresco; Kendro Laboratory Products, Osterodes, Germany) for 2 min. Finally, approximately 100 copies per reaction of an internal positive control (IPC) was included (Table 1). The copy number was calculated from OD260 measurements on the undiluted samples, using the nearest neighbour transformation (see http://www.genosys. com/oligo_uvquant.asp). The speci®city of the IPC was tested with a search in GenBank, as well to the other targets ampli®ed. MQDA-PCR Puri®ed DNA was used in the ampli®cation reactions. We used a two-step PCR ampli®cation approach (Fig. 1). Primers and probes are listed in Table 1. In the ®rst PCR step we used 10 pmol of each of the bipartite primers, 1 3 Dynazyme DNA
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Nucleic Acids Research, 2003, Vol. 31, No. 11 e62 samples with low content of maize DNA. All samples were analysed in duplicate. Sequence-speci®c labelling
Figure 2. Positions of primers and probes in the different GM maize constructs. Approximate positions of primers are shown as arrows, horizontal bars show probe positions. M, MQDA-PCR; T, 5¢ nuclease PCR. Promoters are shown in grey, ®lled circles indicate speci®c point mutations in the epsps gene. P-35S, sequence derived from cauli¯ower mosaic virus promoter; hsp70, sequence containing intron no. 1 of the 70 kDa heat-shock protein of maize; IVS2, intron from maize alcohol dehydrogenase; cryA(b), synthetic delta endotoxin gene derived from Bacillus thuringiensis; T-nos, transcription terminator from Agrobacter tumefaciens nopalin synthase gene; pat, phosphinotricin N-acetyl transferase gene from Streptomyces viridochromogenes; pepC, phosphoenolpyruvate carboxylase promoter; pepC#9, fragment containing intron no. 9 from maize phosphoenol pyruvate carboxylase; T-35S, terminator from cauli¯ower mosaic virus; Pr-act, rice actin promoter; OTP, optimised transit peptide sequence; m-epsps, point mutated epsps gene derived from maize; cryIA(c) and cry9c, delta endotoxin genes from B.thuringiensis.
polymerase II reaction buffer, 0.2 mM dNTP (Promega, Madison, WI), 2 ml of Dynazyme DNA polymerase II (2 U/ml; Finnzymes Oy, Espoo, Finland), and 5 ml of puri®ed DNA in a ®nal volume of 50 ml. For the Bt11-speci®c amplicon, the concentration of primers was increased to 20 pmol (3). The ampli®cation protocol used was as follows (®rst PCR step): four cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s. Twenty microlitres of the ampli®cation product from the ®rst PCR was treated with 20 U exonuclease I (U.S. Biochemical Corp., Cleveland, OH) to degrade the residual single-stranded primers, and 4 U shrimp alkaline phosphatase (U.S. Biochemical Corp.) to inactivate nucleoside triphosphates. The reaction was incubated at 37°C for 30 min, and then at 95°C for 10 min to inactivate the added enzymes. Five microlitres of the treated products was then used for the second PCR ampli®cation. Fifty pmoles of a universal primer identical to the universal HEAD region of the primers used in the ®rst PCR was added. The other components were the same as in the ®rst ampli®cation. The second PCR step was carried out under the following conditions: 30±40 cycles of 95°C for 30 s and 70°C for 45 s. When the system was used to quantify unknown samples, reference samples of known concentrations of GM maize were always included in the same experiment. The ratio of the speci®c signals over the maize reference signal were normalised against the averaged signal from the maize reference gene and used to construct the normalised standard curves. The internally added synthetic DNA, the IPC control, was used as a control for the activity of the DNA polymerase and to identify
After ampli®cation with the HEAD primer the ampli®cation products were treated with 20 U exonuclase I and 4 U shrimp alkaline phosphatase at 37°C for 30 min to degrade residual oligonucleotides and to dephosphorylate trinucleotides, and then 95°C for 10 min to inactivate the enzymes. The cyclic labelling conditions were as follows: 13 Thermosequenase reaction buffer, 10 pmol of each GM-speci®c probe, 100 pmol ddNTP (except ddCTP) (Roche Biochemicals, Mannheim, Germany), 100 pmol ¯uorescein-12-ddCTP (Perkin Elmer, Boston, MA), 16 U Thermosequenase DNA polymerase (Amersham Pharamacia plc, Buckinghamshire, UK) and 24 ml of phosphatase and exonuclease treated PCR product in a total volume of 60 ml. The labelling was done using the following conditions: 95°C for 15 s, 60°C for 1 min for 15 cycles, 95°C for 15 s, 55°C for 1 min for 15 cycles, and ®nally 95°C for 15 s, 50°C for 1 min for 15 cycles. DNA array hybridisation Fifty pmoles of probes complementary to those used in the labelling reaction were spotted on Gene screen Plus nylon membranes (Perkin Elmer), and crosslinked for 15 min with a UV transilluminator (Model TL33; UVP Inc., San Gabriel, CA). The membranes were prehybridized in 0.5 M Na2HPO4 pH 7.2 and 1% SDS for 2 h. The labelled probes were added to 300 ml of 13 SSC and 6% PEG 1500 and heated to 80°C for 5 min. The hybridisation was done overnight at room temperature with agitation in a Cross Blot Dot Blot hybridisation chamber (Sebia, Moulinaux, France) using wells perpendicular to those employed when spotting the complementary probes. The membrane was subsequently rinsed in 13 SSC, 1% SDS for 5 min, then 5 min in 0.13 SSC, 0.1% SDS, and ®nally 5 min in 0.1 M Tris±HCl pH 7.5 and 0.15 M NaCl (antibody buffer). At this point the ¯uorescence was detected directly using a confocal laserscanner (Typhoon Variable Imager; Amersham-Pharmacia). The membranes were then blocked for 1 h in blocking buffer: antibody buffer containing 1% skimmed milk (Difco, Detroit, MI). Blocking buffer containing 1/500 anti¯uorescein horse radish peroxidase (HRP) conjugate was then added, and the reaction continued at room temperature for 1 h. Finally, the membranes were rinsed for 30 min in antibody buffer, and the signals detected with 4 CN Plus chromogenic substrate according to the manufacturer's recommendations (Perkin Elmer). Quanti®cations of both ¯uorescent and chromogenic signals were carried out using the ImagemasterÔ Array software version 2.0 program and calculations were done with Microsoft Excel 97 SR-2 (Microsoft Corp., Redmond, WA). 5¢ Nuclease PCR 5¢ Nuclease PCR was carried out essentially as described (15) using the primer probe systems described in Table 1 and the Applied Biosystems 7700 Sequence Detector (Applied Biosystems).
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Table 1. Primers, probes and synthetic template DNA used in MQDA-PCR and 5¢ nuclease PCR Template
Orientation
Name
Sequence (5¢ to 3¢)
H Mon810F1101MH Mon810R1101MH Mon810 Mud1101 AHJ-2MH Bt11 RBMH Bt11 MudF PepC-20MH Cry04(SMT-CT96)MH Bt176-cryA1-1904 T25 FMHB T25 RMHB T25 Mud GA21 FMHB GA21 RMHB GA21 MUDR GA21 MUD DBT418 FMHB DBT418 RMHB DBT418 MUDR CBH351 FMHB CBH351 RMHB CBH351 MUD 35SH-1 35SMHH-R2 35S Mud2F Ampres FMH Ampres RMH Amp pro Nos FMH Nos RMH Nos pro IPC-FMH IPC-RMH IPC pro ZM1 FMH ZM1 RMH MudF
TGC TAT GCG CGA GCT GCG H-AAT AAA GTG ACA GAT AGC TGG GCA H-CCT TCA TAA CCT TCG CCC G ACG AAG GAC TCT AAC GTT TAA CAT CCT TTG C H-CGC ACA ATC CCA CTA TCC TT H-GCC TCC CAG AAG TAG ACG TC AAG AAA CCC TTA CTC TAG CGA AGA TCC T H-ATC TCG CTT CCG TGC TTA GC H-GGT CAG GCT CAG GCT GAT GT TGA GCA ACC CCG AGG TGG AGG TG H-CCA GTT AGG CCA GTT ACC CAG A H-TGG GAA CTA CTC ACA CAT TAT TAT AGA GAG AGA CTG GTG ATT TCA GCG GGC ATG H-AGC CTC GGC AAC GTC AGC H-TCT CCT TGA TGG GCT GCA G AAG GAT CCG GTG CAT GGC CGG GCC GGC CAT GCA CCG GAT CCT T H-GTC ATT TCA GGA CCA GGA TTC AC H-CCT CTA TTC TGG ATG TTG TTG CC GAA GAA TTC AGC CTA ACC AAG TCG CCT C H-GGT CAG ATC GTG AGC TTC TAC CA H-CGC ATG AAA GCT TCC CAG AT GCT GAA CAC CCT GTG GCC AGT GAA H-GCT CCT ACA AAT GCC ATC A H-CTT GCT TTG AAG ACG TGG TTG G TGC CGA CAG TGG TCC CAA AGA TGG A H-TGC TCA CCC AGA AAC GCT G H-TTC TTC GGG GCG AAA ACT CTC GTA AAA GAT GCT GAA GAT CAG TTG GGT GCA H-GAA TCC TGT TGC CGG TCT TG H-AAT TTA TCC TAG TTT GCG CGC TA TTT ATG AGA TGG GTT TTT ATG ATT AGA GTC CCG H-CGC AGC GTT TCA AGC AGC H-CCA GTT AGC GGG CAG TAT CG AGC AGA CGG TAC GAT CAG ACG CTG T H-TTG GAC TAG AAA TCT CGT GCT GA H-GCT ACA TAG GGA GCC TTG TCC T CAA TCC ACA CAA ACG CAC G
Primers and probes for the MQDA-PCR HEAD sequence Mon810 (15) Bt11 Bt176 T25 (19) GA21 (19)
DBT418 CBH351 35S Amp Nos IPC ZM ref
Sensea Antisensea Probeb Sense Antisense Probe Sense Antisense Probe Sense Antisense Probe Sense Antisense Probe Probe capture Sense Antisense Probe Sense Antisense Probe Sense Antisense Probe Sense Antisense Probe Sense Antisense Probe Forward Reverse Probe Sense Antisense Probe
Primers and DNA used for template construction T25 T25 1±5¢ T25 1±3¢ GA21 GA21 F GA21 R DBT418 Template DBT418hele CBH351
Template
CBH351hele
35S
Sense Antisense Forward Reverse Template
P35S 1±5¢ T35S 1±3¢ IPC-F IPC-R IPC-T
IPC
5¢ nuclease PCR primers and probesc Bt11 Sense Antisense Probe Bt176 Probe T25 Sense Antisense Probe DBT418 Sense Antisense Probe CBH351 Probe zein (20) Sense Antisense Probe aAll
Fbt11-enhpatjun-AHJ-1 Rbt11-enhpatjun-AHJ-2 Fam-Bt11-enh-patd Bt176-CryA1td T25 1±5¢ T25 1±3¢ T25 prod DBT418 F DBT418 R DBT418 prod CBH351 prod Zetm1 Zetm3 Zetmpd
GCC AGT TAG GCC AGT TAC CCA TGA GCG AAA CCC TAT AAG AAC CCT AGC CTC GGC AAC GTC AGC TCT CCT TGA TGG GCT GCA G GTC ATT TCA GGA CCA GGA TTC ACT GGA GGC GAC TTG GTT AGG CTG AAT TCT TCC GGC AAC AAC ATC CAG AAT AGA GG GGT CAG ATC GTG AGC TTC TAC CAG TTC CTG CTG AAC ACC CTG TGG CCA GTG AAC GAC ACC GCC ATC TGG GAA GCT TTC ATG CG ATT GAT GTG ATA TCT CCA CTG ACG T ACT AAG GGT TTC TTA TAT GCT CAA CA CGC AGC GTT TCA AGC AGC CCA GTT AGC GGG CAG TAT CG CGC AGC GTT TCA AGC AGC ACA TCA TCG ATC TAA TCG AGC AGA CGG TAC GAT CAG ACG CTG TCA TAC GCA TAA TCG ATA CGC GAT ACT GCC CGC TAA CTG G CTT GGC GGC TTA TCT GTC TC GCT GCT GTA GCT GGC CTA AT TCG ACA TGT CTC CGG AGA GGA GAC C CTG AGC AAC CCC GAG GTG GAG GT GCC AGT TAG GCC AGT TAC CCA TGA GCG AAA CCC TAT AAG AAC CCT GCA TGC CCG CTG AAA TCA CCA GTC T GTC ATT TCA GGA CCA GGA TTC AC CCT CTA TTC TGG ATG TTG TTG CC GGA GGC GAC TTG GTT AGG CTG AAT TCT TC TGC TGA ACA CCC TGT GGC CAG TGA TGT TAG GCG TCA TCA TCT GTG G TGC AGC AAC TGT TGG CCT TAC ATC ATC ACT GGC ATC GTC TGA AGC GG
sense and antisense primers used in the MQDA-PCR contain the HEAD sequence, designated by an H at the 5¢end in addition to the given sequence. ®lter-bound capture probes are complementary to their corresponding probes. cOnly primers and probes different from those used in the MQDA-PCR are listed. All 5¢ nuclease primers are without the HEAD sequence. dAll 5¢ nuclease PCR probes contain 5¢ FAM (6-FAM) and 3¢ Tamra. bAll
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Nucleic Acids Research, 2003, Vol. 31, No. 11 e62
Figure 3. Eight-plex quantitative determinations of the Mon810 maize in the absence (A) and presence (B) of 2% Bt11 maize together with quanti®ed ¯uorescence signals (C). Each column represents one MQDA-PCR, each row represents one amplicon. Samples: 1, 2: 2.0% Mon810 maize; 3, 4: 1.0% Mon810; 5, 6: 0.5% Mon810; 7, 8: 0.1% Mon810; 9, 10: 0% Mon810; R, reference sample containing 0.7% of each of Mon810, Bt11 and Bt176 maize. N, no template control. The HRP-enhanced chromogenic signals are shown. (C) Quanti®cation of the ¯uorogenic signals of Mon810 alone (diamonds) and Mon810 together with 2% Bt11 (squares). All samples were analysed in duplicate. Amp, ampicillin-resistance gene; Nos, T-nos transcription terminator from A.tumefaciens nopalin synthase gene; 35S, P-35S sequence derived from cauli¯ower mosaic virus promoter; ZM ref, sequence derived from the hmga gene endogenous to maize.
RESULTS AND DISCUSSION The MQDA-PCR was thoroughly evaluated in an eight-plex quantitative detection of the GM maize constructs Mon810, Bt11 and Bt176, the hmga reference gene, the IPC, in addition to the common GM elements 35S promoter and nos terminator (Fig. 3). A series of samples containing dilutions of Mon810, alone or with 2% Bt11 DNA, were analysed. In all cases, samples containing Mon810 gave a linear response proportional to the GM content in the range 0.1±2.0%. The linear regression curves gave squared regression coef®cients of 0.987 and 0.974 for the samples without and with Bt11, respectively. For the samples containing only Mon810 the Pearson correlation between the Mon810 and the 35S signals was 0.994. The 35S signals from the samples containing both Mon810 and Bt11 gradually weaken as the amount of Mon810 is lowered and approach a constant value due to presence of Bt11 DNA. The Bt11, nos and IPC signals (Fig. 3B) remained constant with average values 1900 6 580, 8100 6 230 and 2780 6 960, respectively, and were not affected by the Mon810 content. We also analysed sample series containing Bt11 and Bt176. These samples gave good linear responses in the range 0.1±2% GM material, with square regression coef®cients of 0.998 and 0.978, respectively. Mixtures of Mon810, Bt11 and Bt176 were also analysed. The signals for each target remained unchanged or could be slightly weaker (20±30%) when more than one target was present in the same sample, compared to the signals obtained for single targets (results not shown). This is probably due to a slightly higher frequency of side reactions when multiple targets are ampli®ed simultaneously (see also discussion for twelve-plex PCR). To investigate the in¯uence of template DNA concentration on quanti®cation, a reference mixture of 0.7% of each of Mon810, Bt11 and Bt176 at different dilutions was used as template in the PCR. The undiluted sample contained approximately 100 (determined from 5¢ nuclease PCR) genome copies of the respective GM constructs. The sample was diluted in 4-fold dilutions up to 64-fold. The signals were relatively stable up to a 16-fold dilution. For the more diluted
samples, however, there were probably stochastic effects due to the few target copies. The multiplex system was expanded from an eight-plex to a twelve-plex PCR through the inclusion of primers for detection of the maize constructs CBH351, DBT418, GA21 and T25 (Fig. 4A). Mixtures containing 0.7 or 1.0% of each of all seven different GM constructs were ampli®ed in one reaction together with the amplicons from amp, nos, 35S, IPC and the maize reference gene. When CBH351, DBT418, GA21 and T25 were ampli®ed separately, a dose response was observed. Speci®c signals were obtained for all the constructs. The signals from the mixtures of the seven GM constructs were generally weaker than those obtained with single GM samples (Fig. 4B). The signals obtained for CBH351 in the mixture was ~40% of the signal obtained for CBH351 alone. The corresponding values for DBT418, GA21 and T25 were 35, 35 and 55%, respectively. It is likely that the reduced signals are due to unspeci®c side reactions since all the targets are affected in the same manner. An inherent property of all PCRs is unspeci®c side reactions. Side reactions are not completely eliminated in the twelve-plex PCR, although the MQDA-PCR drastically reduces this problem (see example below where the bipartite primers are not removed after the ®rst PCR). The problem could probably be reduced further by extensive primer optimisation of the bipartite primers. Another option could be to use experimental design to include standards that cover the expected variation. With two level fractional factorial design (0.1 and 1% of the respective GM constructs) quanti®cations of unknown samples should be possible with eight standards per analytical series for the seven GM constructs (determined with the experimental design software Design-Expert version 6.0.7.; Stat-Ease Inc, Minneapolis, MN). In some cases, the standard curves deviate from linearity. This is particularly observed when signals are strong and stems from saturation of targets during the probe labelling reaction. This may be adjusted by lowering the number of labelling cycles. Seventeen different food and feed samples were screened using both the twelve-plex MQDA-PCR and simplex 5¢ nuclease PCRs for the respective GM constructs. Ct values for
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Figure 4. Twelve-plex system for detection of seven different GM maize events. (A) HRP-enhanced chromogenic signals and (B) quanti®cations of the corresponding ¯uorogenic signals for CBH351, DBT418, GA21 and T25. Samples 1, 2: a mixture of 0.7% of each of Mon810, Bt11 and Bt176 and 1% of each of T25, GA21, CBH351 and DBT418; 3, 4: non-GM maize; 5, 6: 2.0% CBH351; 7, 8: 0.5% CBH351; 9, 10: 2% DBT418; 11, 12: 0.5% DBT418; 13, 14: 2% GA21; 15, 16: 0.5% GA21; 17, 18: 2% T25; 19, 20: 0.5% T25. Amplicons are as described in the legend to Figure 3 with additions of amplicons for CBH351, DBT418, GA21 and T25 as indicated in Figure 2. (Squares) Signals obtained from samples 1 and 2.
the maize reference gene in the food and feed samples were generally in the range 26±29. This corresponds to approximately 104±105 gene copies. Ten samples were GM positive, and seven GM negative (GM content 2 >2 >2
