(Transgênicos) Rönning et al., 2003. Event specific real-time quantitative PCR for genetically...

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Eur Food Res Technol (2003) 216:347354DOI 10.1007/s00217-002-0653-4

O R I G I N A L P A P E R

Sissel B. Rnning Marc Vatilingom Knut G. Berdal Arne Holst-Jensen

Event specific real-time quantitative PCRfor genetically modified Bt11 maize (Zea mays)

Received: 8 September 2002 / Revised: 4 November 2002 / Published online: 16 January 2003 Springer-Verlag 2003

Abstract We report the characterisation of the 3-integration junction between host plant DNA and inte-grated gene-construct of the genetically modified Bt11

maize, and the successive development and first valida-tion of a transformation event-specific quantitative Taq-Man 5-nuclease PCR assay for detection and quantitationof Bt11 maize on the LightCycler. The transgenic DNA inBt11 is inserted in a tandem repeated DNA sequencemotif of approximately 180 bp, and we discuss some ofthe problems this creates for design, development andvalidation of event-specific PCR assays. The limit ofdetection of the PCR assay was estimated to 10 initialtemplate copies. The limit of quantitation in pure maizematerials was estimated to approximately 40 templatecopies. In food samples containing high levels ofexogenous DNA, the impact of this DNA was estimated

to increase the limit of quantitation to approximately 100initial template copies. Because of the low number oftemplate copies required for detection and quantification,in combination with small size of the amplicon (~100 bp),the assay may be applied to analyses of raw materials aswell as processed food and feed.

Keywords Event-specific PCR Real-time PCR Genetically modified organism GMO Bt11 maize Detection and quantitation limit Integration site

Introduction

At the present time, 19% of the worlds planted maize(Zea mays) acreage is genetically modified (GM) [1].

Consumers are concerned about the use of geneticallymodified plants in food production, and as a consequence,labelling of GM food has become an important issue. The

principles of European legislation on GM foods, as laiddown in EC regulations: 258/97 [2], 1139/98 [3], 49/2000[4] and 50/2000 [5], is that a product shall be mandatorylabelled if it consists of a GM organism (GMO) or if it isderived from a GMO but no longer contains the GMO andthere is still modified DNA or protein present. The legalthreshold value for labelling, set down by the EuropeanCommissions Standing Committee in 1999, is 1%.

Methods to detect GMO-derived DNA can be dividedinto four categories; screening, gene-, construct- andevent-specific, and their ability to discriminate betweenGM- and non-GM-derived DNA varies accordingly [6].The screening methods are associated with a particular

risk of yielding false positives [7]. The methods fordetection of the modified genes are somewhat morespecific, although the modified genes may be used inseveral GMO and with variable copy number, hencereducing the specificity and accuracy for quantitation.Construct-specific methods are normally specific forGMO, and the risk of reporting false positives issignificantly lower than with screening and gene-specificmethods. However, construct-specific methods cannotdistinguish between different GMO if the same constructhas been integrated. Furthermore, the number of integrat-ed copies of the PCR target sequence may vary betweendifferent GMO containing the same target sequence. Theintroduction of new GMO in the future, containing manyof the same genetic elements, will reduce the specificityof the construct-specific methods. For example, Mon809and Mon810 maize have been transformed with the sameplasmid, PV-ZMBK07, containing a PE35Shsp70in-tronCryIA(b)Tnos construct [8].

To overcome specificity problems, a line or transfor-mation event-specific (hereafter termed event-specific)PCR may be performed. Since, with currently usedtransformation techniques, the integration of an intro-duced gene-construct into the genome of a plant isunlikely to occur twice at the same genomic locus, the

S. B. Rnning K. G. Berdal A. Holst-Jensen ())Section of Food and Feed Microbiology,National Veterinary Institute,Ullevlsveien 68, P.O. Box 8156 Dep., 0033 Oslo, Norwaye-mail: [email protected].: +47 23 21 62 43Fax: +47 23 21 62 02

M. VatilingomTEPRAL, 68 route dOberhausbergen, 67200 Strasbourg, France


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junctions between the integrated DNA sequence and thehost DNA are unique for a single transformation event.The use of a PCR system targeting this integration borderwill be event-specific because the assay is specific forboth the transgenic construct and the integration site.Furthermore, since a single locus is targeted, event-specific methods (in contrast to other methods) will havefew copy number variation problems. Until now, event-specific quantitative methods have been published forRoundup Ready soybean (RRS) [9, 10, 11] and Mon810maize [12] using real-time PCR, and for Bt11 maize [13]using competitive PCR.

A major portion of the transgenic crops will bemarketed as processed products (e.g. meat mixed with soyprotein from GM soybeans). The applicability of the PCRmethods for GMO detection in processed foods isdetermined by the quality of the DNA still present inthe final product. Physical and chemical parameters maylead to random cleavage and/or removal of the genomicDNA. With increased processing, the size of the genomicDNA-fragments decreases. Consequently, the ampliconsize in an analysis should be minimised to obtain thehighest achievable detectability. This will make it moredifficult to detect the presence of GMO with conventionalagarose gel electrophoresis. Furthermore, quantitation of

the amplified DNA is difficult if image analysis iscombined with agarose gel visualisation and smallamplicons. To overcome the size problem, a DNAhybridisation technique is commonly applied for detec-tion, identification and quantitation [9, 10, 11, 12, 14, 15].

This paper describes the characterisation of the3event-specific integration junction of Bt11 and thesuccessive development and first validation of primersand TaqMan probes [16] for quantitative real-time PCRassays targeting the event-specific upstream and down-stream integration junctions of Bt11 on a LightCycler

(Roche) real-time thermal cycler. The maize endogenousinvertase gene is used as a reference gene.

Materials and methods

Characterisation of the 3-integration junction

The 3-integration junction was characterised using a modifiedversion of the inverse PCR protocol originally described by Hartl etal [17].

Digestion and ligation of genomic DNA

Consejo Superior de Investigaciones Cientficas (CSIC, Barcelona,Spain) provided CsCl2 purified and ethanol precipitated DNA from100% Bt11 homozygous leaf material for the characterisation of the3-integration junction. Genomic Bt11 DNA (1.5 g) was digestedusing 20 units of EcoRI, XhoI and HindIII (Promega) for 3 h at37 C in a total volume of 50 L in three separate tubes, andsuccessively purified on a silica gel column (Qiagen). One volumeof digested DNA was added to 5 volumes of PB buffer (Qiagen),transferred to the spin column and centrifuged for 1 min at15 000g. After washing twice with 700 L of PE buffer, the DNAwas eluted with 100 L of EB buffer (10 mM Tris-HCl, pH 8.0).

The digested DNA was self-ligated [17] in order to get smallcircularised genomic DNA as follows: ten units of T4 DNA ligase(Promega) was used to ligate 500 ng of digested DNA in a totalvolume of 500 L for 24 h at 4 C. The DNA was purified as

described above using a Qiaquick DNA cleanup kit (Qiagen), andeluted in a final volume of 50 L.

Inverse PCR

The ligated product was amplified using a modified inverse PCRprotocol [13] to obtain enough material for sequencing. InversePCR reactions were performed on a GeneAmp 2400 thermal cycler(Applied Biosystems), in 50 L final volume, containing 1xreaction buffer (Qiagen), 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 M ofeach primer and 2.5 units of Taq DNA polymerase (Qiagen) in anested approach. The first amplification contained 2 L ofpreviously purified ligated DNA and the primers Pat/Bar5 and

Table 1 Primers and probes used for characterisation of the 3integration- junction and for real-time PCR quantitation of Bt11 content

PCR assay Primer/probea Name Sequence of oligonucleotide 5fi3 Size (bp)

Primers for characterisation of Bt11

Inverse PCR Forward Pat/Bar3 CCA GTT AGG CCA GTT ACC CAG ATFirst PCR Reverse Pat/Bar5 AAA CCT TGC GCC TCC ATA GAC TTInverse PCR Forward Nos2 TGA TTA GAG TCC CGC AAT TAT ~1 kb2 kbNested PCR Reverse Pat/Bar1 ACA TT AAC CTT TGA TGC CTA TGT G

Reference gene primers and probe for real-time PCR

Maize invertase gene Forward ZmayInvFor CAC TCC ATC GTG GAG AGC TT 111Reverse ZmayInvRev GGC GTT GTT GAA GAG GAA GAProbe ZmayInvFT TAC CCC ACA CGA GCC ATC TAC GAC T

Bt11 specific primers and probes for real-time PCR

Bt11 3 junction Forward Bt113JFor GCG GAA CCC CTA TTT GTT TA 70Reverse Bt113JRev TCC AAG AAT CCC TCC ATG AGProbe Bt113JFT AAA TAC ATT CAA ATA TGT ATC CGC TCA

Bt11 5 junction Forward Bt111b TAT CAT CGA CTT CCA TGA CCA 82Reverse Bt115JRev CTT TGA TCT TTT CTA CGG GGT CTProbe Bt115JFT TTT TCG TTC CAC TGA GCG TCA GAC C

a The probes were labelled with 5-FAM and 3-TAMRAb With exception for primer Bt111 developed by Zimmermann et al [13], the primers and probes were developed as part of this study

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Pat/Bar3 (Table 1), using a PCR program consisting of a first 95 Cdenaturation step for 2 min followed by 25 cycles of 30 sdenaturation at 95 C, 30 s annealing at 56 C and 1 min 30 sextension at 72 C. The second (nested) amplification contained2 L of the amplification product from the first PCR reaction andthe primers Pat/Bar1 and Nos2 (Table 1), using a PCR programconsisting of a first 95 C denaturation step for 2 min followed by35 cycles of 30 s denaturation at 95 C, 30 s annealing at 52 C and1 min 30 s extension at 72 C.

All the PCR products were separated by electrophoresis on

1.5% agarose gel (TBE buffer). The fragments were successivelyisolated from the gel by cutting the gel under UV-light andpurifying using Qiaquick Gel extraction kit (Qiagen). The frag-ments were cycle sequenced with Big Dye terminator v.3 (AppliedBiosystems) on an ABIprism 310 (Applied Biosystems).

Design and validation of a detection assays

Maize DNA samples and extraction

DNA was extracted from certified reference materials (CRMs)produced by the Institute for Reference Materials and Measure-ments (IRMM, Geel, Belgium) and commercialised by Fluka, usinga CTAB-based protocol [18]. The Bt11 CRMs (IRMM-412) are amixture of dried heterozygous Bt11 grain and non-GM maize,

produced on a weight:weight basis.CSIC provided CsCl2 purified and ethanol precipitated DNAfrom 100% Bt11 homozygous leaf material for the construction ofcalibration curves. In addition, DNAs extracted from CRMs ofBt11 (IRMM-412), Bt176 (IRMM-411), Mon810 (IRMM-413) andRRS (IRMM-410R), and from the cloning vector pUC18 (Amers-ham Biotech) were used as templates in order to validate theTaqMan assays. DNA concentrations were measured in ng/Lusing a SYBR Green method [9].

Primers and probes

Different primer sets with corresponding probes were designed forthe 3-integration junction between the host DNA and the insertedgene construct. In addition, the event-specific detection system for

the 5-junction described by Zimmermann et al. [13] was modifiedto fit a real-time PCR assay by designing a TaqMan probe and newreverse primers. The primers and probes were designed using thesoftware program OLIGO-Applet [19], specifying an optimalmelting temperature (Tm) of 60 C for the primers, and 70 Cfor the probes. The full-length maize endogenous invertase gene[20] (EMBL/GenBank accession number U16123) was used asinput sequence for the development of primers and a correspondingprobe for a reference gene. The probes were labelled with 5-FAMand 3-TAMRA. All the primers and probes were purchased fromDNA Technology (rhus, Denmark).

PCR conditions

The PCR reaction volume of 30 L contained 5 L template DNA,0.75 M of each primer, 0.25 M probe, 1.5 units Taq DNApolymerase (AmplitaqGold, Applied Biosystems), 0.3 units uracil-N-glycosylase (AmpErase UNG, Applied Biosystems), 0.1 mg/mLBSA, 0.2 mM dATP, dCTP and dGTP (Applied Biosystems) and0.4 mM dUTP (Applied Biosystems), 4 mM MgCl2 and 1x PCRbuffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl). The event-specific target sequence and the maize endogenous invertase genewere amplified in separate capillaries, and the fluorescence wasmeasured in channel 1 of the LightCycler and analysed withsoftware version 3.5. The PCR-program included an initialdecontamination step for 2 min at 50 C to allow optimal UNGenzymatic activity, followed by 10 min at 95 C in order to activatethe DNA polymerase, to deactivate the UNG and to denature thedouble-stranded template; and successively 50 cycles of 15 s

denaturation at 95 C and 30 s synthesis at 60 C. The samereaction conditions and solutions were used for the maizeendogenous invertase gene.

Calibration curves

The haploid genome size of maize has been estimated to be withinthe range of 22922716 Mbp, corresponding to a molecular weightof ~2.5 pg for the haploid genome [21]. Calibration curves for the

amplicons were established with fourfold dilutions of DNA from100% Bt11 material (containing approximately 20 000 to 5template copies per PCR reaction).

Estimation of the limit of detection (LOD) and the limit ofquantitation (LOQ)

To determine the LOD and LOQ, 2% Bt11 CRM (IRMM-412) wasused. Due to heterozygosity this material contained approximately100 copies of the maize genome per copy of the Bt11 event-specifictarget sequence. The DNA was diluted in a fourfold dilution seriesand each concentration was analysed in four parallels. In order toestimate the impact of exogenous background DNA and to mimicthe situation in real food samples, a background of 20 000 soybeangenome equivalents (IRMM-410R) per 5 mL template DNA was

added to each of the dilutions of the 2% Bt11 CRM.

Results and discussion

Bt11 transgene 3-integration junction characterisation

In order to obtain the Bt11 integration 3-junctionfragment, a modified inverse PCR method based on the5-junction characterisation method described by Zim-mermann et al. [13] was used. The inverse PCR primersannealed between the Pat/Bar gene and the NOS termi-nator (Fig. 1). After the first amplification reaction with

the primers Pat/bar3 and Pat/bar5, no PCR products weredetectable with agarose gel electrophoresis. Consequent-ly, a nested PCR with 35 cycles was performed using theprimers Pat/bar1 and Nos2. Two PCR products ofapproximately 1 and 2 Kb length were amplified withEcoRI and XhoI digested DNA, respectively (Fig. 2).With HindIII digested DNA, no amplification productwas obtained. The two fragments were sequenced andwere 100% homologous. A part of the consensusalignment including the transgene 3-integration junctionis shown in Fig. 3. The consensus sequence has beensubmitted to EMBL/GenBank under accession numberAY123624.

Development of real-time PCR assays

Different primer sets for both the 5- and 3-junctionswere subjected to a first qualitative PCR test using DNAextracted from 2% Bt11 CRM. For each junction, theprimer pairs that produced the single band with highestintensity were selected to develop a quantitative Bt11real-time PCR assay (Table 1). A map of the Bt11 5- and3-junctions and the location of the primers and corre-sponding probes is shown in Fig. 3. We selected primer-

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sets yielding amplicons of 111 bp for the endogenousinvertase gene, and 82 bp and 70 bp for the 5- and 3-event-specific junctions, respectively. For all the PCRreactions, the same thermal PCR program was used. Inorder to optimise the PCR conditions we varied theMgCl2 concentration within the range of 26 mM. Forboth the 5- and 3-junction assays, 4 mM MgCl2 yieldedthe best amplification (lowest Ct values). Further optimi-sation and testing indicated that the 5-assay was lessspecific than the 3-assay. The 3-assay was thereforechosen as our primary target for further design andvalidation of a real-time quantitative assay.

Calibration curves

Two parallels of each fourfold dilution (corresponding toapproximately 20 000 to 5 genome equivalents of 100%Bt11 DNA per PCR) were used to establish calibrationcurves for the invertase and the 3-assay (Fig. 4). Thesquare regression coefficients (R2) were 0.9973 and0.9986 for the invertase and the Bt11 amplicons, respec-tively. Good linearity between copy number and fluores-cence values (Ct) visualised in the calibration curves inFig. 4, i.e. high agreement between the amount of

template (ln input DNA) and amount of product (Ctvalues), indicates that this assay is well suited forquantitative measurements.

Accuracy and specificity of the method

In order to determine the accuracy (precision andtrueness) of the quantitative 3-assay we used DNAextracted from CRMs of relative concentrations of 0, 1.0and 2.0% Bt11 as template in the PCR reactions. Eachtemplate was analysed in five parallels, and the quanti-tative estimates were computed using standard curvesmade from dilution series of known amounts of invertaseand Bt11. No amplification signal was observed withDNA from 0% Bt11, while estimates from 1% Bt11 CRMranged from 0.71% to 1.21% with a mean of 0.99% and aS.D. of 0.22%, and estimates from 2% Bt11 CRM ranged

Fig. 2 Inverse PCR amplification results with Bt11 DNA digestedwith EcoRI (E1 to E6, black arrow) and XhoI (X1 to X6, whitearrow). Six independent parallel experiments were performed foreach enzyme (E1-E6 and X1-X6). L Low mass ladder (GibcoBRL), N PCR negative control

Fig. 3 Sequence of the 5- and 3-integration junction regions andlocation of the primers and the TaqMan probes for the event-specific amplification and detection of Bt11 maize. The asterisksand arrows indicate the exact junctions between the host-plantgenomic DNA and the inserted DNA, and the amplicons arepresented as double-stranded sequences in bold letters. The namesand the direction of the primers (normal types) and probes (italics)are indicated. The maize endogenous DNA sequences are shown insmall italics, while the insert DNA is shown in capitals. Top: The5-junction amplicon and the location of the primers and probe. Theupstream primer (Bt111) spans the event-specific 5-junction and

is partially derived from the plant DNA (11 nt) and partially fromthe insert sequence. Bottom: The 3-integration junction andlocation of the primers and probe. The downstream primer(Bt113JRev) spans the event-specific 3-junction and is partiallyderived from plant DNA (9 nt) and partially from insert sequence

Fig. 1 Schematic representation of the Bt11 transgenic map. Theprimers used for the 3 junction characterisation are indicated witharrows. Primers for the first PCR (Pat/bar3 and Pat/bar5) are shown

as open arrows and primers for the second (nested) PCR (Pat/bar1and Nos2) are shown as closed arrows. Reverse primers restrictionsites for EcoRI, HindIII and XhoI are indicated

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Fig. 4AD Amplification andcalibration curves for the maizeendogenous invertase gene andthe Bt11 event-specific 3-junction were established usingdiluted DNA from 100% Bt11material. The dilution seriescontained approximately20 000,5000, 1250, 312, 78, 19 and 4.9initial template copies in the

PCR reactions. A Amplificationcurves for the maize endoge-nous invertase gene, analysedusing Fit Points on the Light-Cycler. B Calibration curvesplotting ln (input DNA) versusCt. C Amplification curves forthe Bt11 event-specific 3-junction, analysed using FitPoints on the LightCycler. DCalibration curves for Bt11plotting ln (input DNA) versusCt

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from 1.13% to 2.22% with a mean of 1.56% and a S.D. of0.48%. The precision (RSDr=0.22) and trueness (er-ror=1.0%) were better for 1% Bt11 CRM than for 2%Bt11 CRM (RSDr=0.31 and error 22%) The DNA fromthe 2% Bt11 we used was extracted from recentlypurchased CRM (June 2001), while the DNA from the1% material was extracted from material purchased inApril 2001. The IRMM has recently performed stability

testing of the CRMs. With Bt11 and Bt176 maize,degradation problems have been discovered on thematerial from May 2001 to February 2002. Consequently,the IRMM-411 and IRMM-412 series were withdrawn inFebruary 2002. DNA degradation, although not sequence-specific, leads to reduction in the amount of extractedDNA and consequently to a reduction in availableamplifiable DNA. Low concentration of the target DNAleads to higher variation among parallel runs than withhigh concentration. This may explain some of therelatively low precision and trueness we observed withthe 2% CRM.

DNA from soybean, Mon810 and Bt176, was used in

order to determine the specificity of both the qualitativeand quantitative 3-assay. With these template DNAs nosignal was detected on the LightCycler after 50 cycles.However, when the reaction products from maize DNAwere analysed on agarose gel, amplification productswere occasionally observed with variable signal intensity.Unspecific amplification products were never observedwhen the PCR was limited to 35 cycles. A homologysearch (blastn) against the EMBL/GenBank database,(http://www.ncbi.nlm.nih.gov/BLAST/), using the 3-event-specific junction sequence of Bt11 as probe,retrieved a maize-specific 180 bp knob-specific repeatwith high homology (EMBL/GenBank accession number

AF030935) [22]. The reason for the observation of falsepositives on agarose gel may be that the Bt11 event-specific primers occasionally amplify parts of thisrepeated region. However, with a TaqMan assay thisproblem is minimised due to the high specificity of theTaqMan probe. The use of a PCR system covering theintegration border, amplifying parts of both the host DNAand the transgenic construct, enables the best and mostspecific PCR system for identification and detection ofGMO. Ideally, an event-specific detection system shallconsist of one primer located in the transgenic constructand one located in the host DNA and an event-specificprobe that spans the junction. The Bt11 gene-construct isintegrated into a maize-specific 180 bp knob-specificrepeat. Consequently, we expected and observed multipleamplification products with gel electrophoresis when aprimer was entirely located in the repeat motif due topriming within several repeats. To overcome theseproblems, the primers designed in our detection systemsspan the junctions, while the probes are placed inside thetransgenic construct. Although, this is not ideal, webelieve this was the best way to obtain high specificity ofthe Bt11 event-specific assay.

The amplicon for the 3-junction assay, with theexception of 9 nucleotides (nt) at the 5-end of the reverse

primer, is 100% similar to pUC18 (EMBL/GenBankaccession number L08752). From this, we predicted thatpUC18-derived amplification products would be generat-ed at least under sub-optimal reaction conditions. In orderto determine the specificity of the assay with respect tothe pUC18 sequence, we used the pUC18 vector as atemplate with the Bt11 event-specific 3-junction assay.As we expected, a positive signal was detected on the

LightCycler when high concentrations of pUC18 wereused as template. There is a significant risk of reportingfalse positives in the presence of concentrations ofpUC18-derived DNA corresponding to the limit ofdetection of pUC18 (1109 template copies with a Ct-value above 40). Such concentrations could be due to thepresence of another GMO containing pUC18 vectorsequences, bacteria transformed with pUC18, or by vectorcontamination of the polymerase. It is normally notpossible to obtain such concentrations of pUC18 in a foodor feed sample, unless it is highly contaminated with e.g.genetically modified microorganisms. Consequently, webelieve this is an unlikely situation in routine analysis. It

is important to realise that a Ct-value >40 usually meansthat the PCR reaction is inhibited or that the target isabsent.

Determination of LOD and LOQ

There are at least three different ways of expressingdetection and quantitation limits, although they all refer tothe lowest quantity of the target that reliably can bedetected and quantified with a probability of 95% [9].The absolute limit is the lowest number of initial templatecopies that can be detected and quantified, the relative

limit refers to the lowest percentage of GMO relative tothe species (e.g. maize) that can be detected andquantified, and the practical limit is the functional limitof the sample during an analysis. In order to determine theLOD and LOQ of the 3-assay, a fourfold dilution seriesof 2% Bt11 CRM DNA containing an estimated averageof 320, 80, 20, 5 and 1.25 copies of the Bt11 genome perPCR were analysed in four parallel real-time PCRanalyses (Table 2 and Fig 5). As expected, the abilityto detect Bt11 decreased with decreasing copy numbers.We were able to detect Bt11 in all the four parallels downto 20 copies, while only three of the parallels werepositive when we used an estimated average of 5 copies,and only one of the parallels was positive when we usedan estimated average of 1.25 copies in the PCR. Theseresults are within the range predicted by a Poissondistribution model and an LOD of approximately 10copies. The lowest number of copies we were able todetect was estimated to 12 initial template copies. Fromthe results, we estimate the absolute LOD to be approx-imately 10 copies of the Bt11 3-junction [9]. Theexperiments show that the Ct variations among parallelsof the same template concentration increase with de-creasing copy number. To obtain reliable quantitationresults under ideal conditions, approximately 40 initial

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template copies are required. Consequently, we concludethat the absolute LOQ with two parallel PCR reactions is40 initial template copies. The estimated LOQ value is,however, only valid under exceptionally optimal condi-tions, i.e. with high molecular size DNA in pure maizesamples.

For real food and feed samples, the LOQ value maydiffer from the estimated LOQ using high concentrationand high purity DNA, e.g. because of large background of

non-target DNA. In order to mimic a situation moresimilar to that in many food and feed samples and toestimate the impact of a high exogenous DNA back-ground on the LOQ value, we made a new dilution seriesby mixing ~20 000 soybean genome equivalents (~20 ng)with approximately 80, 60 and 40 copies of 2% Bt11CRM (Table 3). The large background of soybean-derivedDNA had a major influence when few copies of Bt11were present in the sample (observed values roughly

Fig. 5 Estimation of limit of detection (LOD) and quantitation(LOQ). Amplification curves for Bt11 based on fourfold dilution-series with an estimated average of 320, 80, 20, 5 and 1.25 initialtemplate copies of Bt11 per PCR, analysed using second derivate

maximum on the LightCycler. The Bt11 is detected in all the fourparallels down to 20 template copies, but only in three of fourparallels at 5 copies and one of four parallels at 1.25 copies

Table 2 Amplification dataa used to determine the absolute LOD and LOQ

Estimated template copies Signal rate (#positive signals) Mean Ct-values SD of observed Ct-values

320 4/4 33.22 0.3280 4/4 34.30 0.4520 4/4 36.33 0.73

5 3/4 37.92 1.201.25 1/4 38.63 n.a.

aFourfold dilutions series of 2% Bt11, based on initial template copy numbersn.a. Not applicable

Table 3 Quantitation limit with 20 000 soybean genome equivalents as background in the PCR reaction, using 2% Bt11 and 2% RRS astemplate in the reaction

Initial template copies A B C D Mean SD Influence of backgroundDNA (D Ct)a

80 33.23 33.52 33.71 33.60 33.52 0.2180+Soybean DNA 33.44 33.78 33.67 33.85 33.69 0.18 0.17

60 34.3 33.84 34.04 34.66 34.21 0.3560+Soybean DNA 34.56 34.78 35.09 33.84 34.57 0.53 0.3640 34.31 34.83 34.92 34.77 34.71 0.2740+Soybean DNA 35.19 36.05 34.30 35.56 35.28 0.74 0.57

a Relative to the Ct value observed without soybean DNA in the reaction

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corresponding to the effect of removing approximately1020 copies of Bt11, which with 12,5% of the Bt11 copies). By extrapolation of theobserved effect on Ct-values of adding the soybean DNA,we estimate the effect to be insignificant with 100 copiesor more (cf. Table 2). The fluctuation of the curves wasalso altered by the addition of soybean DNA, yielding asignificant increase in standard deviation with lower copy

numbers and decreasing influence with increased copynumbers. With 80 copies or more, we could no longerobserve this effect. These results correspond to observa-tions we have made with an RRS detection assay [9]under similar conditions. This means that an absoluteLOQ of 40 initial template copies is only representativewith DNA from CRMs or materials of equal or betterquality and with low concentrations of DNA from puremaize materials. However, with a large background ofother DNA, as is typically the case with foodstuffs, higherinitial template copy numbers are required (LOQ isestimated to be approximately 100 copies).

We conclude that the Bt11 maize transformation

event-specific quantitative PCR assay presented here ishighly specific, despite the difficulties we had because ofthe repeated elements at the integration site and theremaining vector sequences in the transgenic DNA. Dueto the small size of the PCR products, this method is alsosuitable for processed food and feed matrices. Theabsolute detection limits and quantitation limits wereestimated to 10 and 100 initial template copies, respec-tively, indicating that the system is highly sensitive.

Acknowledgements We wish to thank Martha Hernandez Perez,CSIC, Barcelona, Spain for providing 100% Bt11 material. Thisstudy was financially supported by the EC FP5 Quality of Life andManagement of Living Resources program within the project

Reliable, standardised, specific, quantitative detection of geneti-cally modified food (contract no. QLK1199901301, acronymQpcrgmofood), and by a grant from the Research Council ofNorway (136430/140). This is gratefully acknowledged.

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