Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed
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- Bahrdt, C., Krech, A.B., Wurz, A. et al. Anal Bioanal Chem (2010) 396: 2103. doi:10.1007/s00216-009-3380-x
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For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LODabs) ≤ ten target copies was proven in hexaplex format. A sensitivity ≤ ten target copies of each GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency.
Each year, an increasing number of different genetically modified (GM) plants are grown on commercial scale. The growing number of different genetically modified crops goes in parallel with an increase in the diversity of genetic modifications in commercialised genetically modified organisms (GMOs). Detection and identification—e.g. to verify compliance with food-labelling requirements—becomes more and more complex. Additionally, GM plants lacking common screening elements enter the food and feed chain and require revision of the common screening procedures. Accidental contamination of seed with ‘non-approved’ GMOs or illegal planting of new varieties (e.g. Bt63 rice) is another issue in this context. Thus broad and at the same time flexible new testing methods are needed. In combination with additional modular identification methods, this allows cost-efficient analysis and gives a comprehensive answer to the question if and which GMO is present in the sample.
Recent approaches consist of an initial amplification step with multiplex polymerase chain reaction (PCR) followed by a method for separation and detection of the PCR products, e.g. by agarose gel electrophoresis with ethidium bromide staining [1–6], capillary gel electrophoresis [7–10] or hybridization microarray technology [11–14].
Another approach is the use of real-time PCR offering a substantial advantage as compared to the former mentioned methods—the lack of post-PCR manipulation. Integration of PCR and detection of the amplification products in a closed system minimises the risk of carryover contamination . Furthermore the capability of real-time PCR for a high automatisation level can reduce hands-on time and consequently the costs per analytical result.
Multiplexing offers advantages as e.g. increased throughput and reduced turnaround times of GMO samples . Furthermore multiplex PCR can increase the reliability by eliminating the risk of intertube variability (e.g. pipetting errors).
Several duplex real-time PCR assays, including cauliflower mosaic virus (CaMV) 35S promoter and/or Agrobacterium tumefaciens NOS terminator PCR detection system, have already been published [17–19]. Lately a quadruplex real-time PCR screening assay has been described comprising PCR systems for the detection of 35S promoter and NOS terminator in combination with reference systems for soy (lectin gene) and maize (alcohol dehydrogenase gene) .
A critical aspect linked to more complex real-time PCR assays and accordingly a higher number of different dyes involved is the increased likelihood of crosstalk. Crosstalk is a fluorescence increase in a detection channel caused by a dye which is not intended to be measured in a given detection channel. Therefore in a multiplex assay, the optimum combination of reporter dyes, which can vary from instrument to instrument, is of particular importance.
Over the past years, mainly new GM maize and GM soybean events have been approved. Here we describe the development and validation of a hexaplex real-time PCR assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay including all currently commercialised GM maize and GM soybean events. Combining three different screening methods, two modification-specific primer sets and an IPC system, the new assay was shown to be time and cost efficient as well as robust and fit for purpose under routine testing conditions. To prove performance, new validation approaches and controls had to be developed and implemented to account for the increased complexity of the multiplexed assay type.
Materials and methods
For specificity testing, certified reference materials (CRMs) and DNA extracts were purchased from IRMM (Geel, Belgium), AOCS (Urbana, IL, USA) and Bayer BioScience (Gent, Belgium) respectively: soybean: GTS 40-3-2 (ERM-BF410gk), MON89788 (AOCS 0906-B), A5547-127 (AOCS 0707-C), A2704-12 (AOCS 0707-B), DP305423 (ERM-BF426d), DP356043 (ERM-BF425d); maize: Bt 176 (ERM-BF411F), GA21 (ERM-BF414d), MON810 (ERM-BF-413-3), Bt-11 (ERM-BF412F), NK603 (ERM-BF415F), MON863 (ERM-BF416D), TC1507 (ERM-BF418D), MIR604 (ERM-BF423D), MON88017 (AOCS 0406-D), 59122 (ERM-BF424D), 3272 (ERM-BF420C), T25 (AOCS 0306-H); rapeseed: T45 (AOCS0208-A), Ms8 (AOCS-0306-F), Rf3 (AOCS0306-G), RT73 (AOCS0304-B), Ms1, Rf1, Rf2, HCN92; cotton: MON1445 (AOCS 0804-B), 281-24-236 × 3006-210-23 (ERM-BF422B); MON531 (AOCS-0804-C), MON15985 × MON1445 (AOCS-0804-D), LLCotton25 (AOCS 0306-E); rice: LLRICE62 (AOCS0306-I); sugar beet: H7-1 (ERM-BF419b); potato: EH92-527-1 (AOCS 0806-D). At the time of development and validation, no CRM for maize LY038 has been available; thus non-certified material had to be used. Non-GM materials tested and used for preparation of DNA mixtures were cotton, soybean, canola, maize, rice, potato and sugar beet.
DNA from GM and non-GM material was extracted by a protocol based on a cetyltrimethylammonium bromide (CTAB) extraction method  including an initial overnight incubation with RNase and Proteinase K. Subsequent purification was performed using gravity flow, anion exchange columns Genomic-tip 500/G (Qiagen, Hilden, Germany) following the instructions of the manufacturer.
Preparation of DNA samples for specificity testing
For specificity testing, 1% GM samples were prepared with a total DNA concentration of 20 ng/µl. Non-GM DNA samples were also normalised to a DNA concentration of 20 ng/µl.
Preparation of positive control material
Positive control material was prepared by cloning PCR products containing the target sequences each into a pCR®2.1 transformation vector (Invitrogen GmbH, Karlsruhe, Germany). Plasmids were then linearised using a restriction enzyme cutting opposite to the multiple cloning site. The plasmid DNA was quantified by fluorescence detection using the Quant-iT™ PicoGreen® dsDNA Reagent (Invitrogen, Karlsruhe, Germany) and diluted with 0.1× TE containing 10 ng/µl ssDNA from salmon testes (Sigma-Aldrich Chemie GmbH, Munich, Germany) to a stock solution of 105 copies plasmid DNA per microlitre, corresponding to 105 PCR target copies per microlitre. These stock solutions were diluted to a working solution of 1,000 copies and used for the preparation of positive control reactions and spiking in development and validation experiments. Asymmetric target ratio samples were also diluted and mixed from these stock solutions.
Primers and probes
For two newly developed construct-specific real-time PCR systems, new primers and TaqMan™ probes have been designed using Primer Express® software v2.0 (Applied Biosystems, Foster City, USA). Primer sequences of the PCR systems: NOS PCR system: forward primer (ggcaataaagtttcttaagattgaatcctg), reverse primer (catgcttaacgtaattcaacagaaatt), probe (HEX–ttgccggtcttgcgatgattatcat–BHQ-1); FMV PCR system: forward primer (aagacatccaccgaagacttaaagttagtg), reverse primer (tctgcaccattccttttttgtctg), probe (CAL610–tgaaagtaatcttgtcaacatcgagcagctgg–BHQ-2); LY PCR system: forward primer (caatctgtgactggtagagggaagg), reverse primer (gccgaagtgctctactccggtctt), probe (Cy5–ttccttggcagccatcactagtacaggttta–BHQ-2); SAMS PCR system: forward primer (gcttgttgtgcagtttttgaagtataacc), reverse primer (gaatcgggtggttctggaa), probe (Cy5–ccacacaacacaatggcggcca–BHQ-2). IPC PCR system: forward primer (agctctttgtgcgaaaggc), reverse primer (gtgaggattcggacacgg), probe (ATTO425–tcgcctcccacgtctcaccga–DDQ-1). The CaMV 35S promoter specific real-time PCR system was taken from the ISO 21570:2005 “Screening method for the relative quantitation of the 35S-promoter DNA of soya bean line GTS40-3-2 using real-time PCR” , but the probe was labelled with FAM as a reporter dye and BHQ-1 as a quencher. ‘In-silico’ multiplex PCR was performed using Clone Manager Professional Version 9 (Scientific & Educational Software, Cary, USA) to evaluate potential oligonucleotide interactions leading to primer/probe dimerisation.
The real-time PCR experiments were performed on Stratagene Mx3005P QPCR system comprising an ATTO425, FAM, HEX, ROX and Cy5 filter set using MxPro–Mx3005p v4.00 Build 367, Schema 80 software (Agilent-Stratagene, Waldbronn, Germany).
Final concentration of the forward and reverse primer in all GMO detection systems was 300 nM and 150 nM for the probes. For the IPC system forward primer, reverse primer and probe were applied with 100 nM. In PCR reactions, a reagent mix from single components has been used containing the following components in specified final concentrations: 1× GeneAmp® PCR Buffer II, 2 units AmpliTaq Gold® DNA polymerase (both from Applied Biosystems, Foster City, USA), 0.01% Tween 20, 0.8% glycerol, 5.5 mM MgCl2 (all from Sigma-Aldrich Chemie GmbH, Munich, Germany) and 200 µM dNTP (GE Healthcare, Munich, Germany).
The total reaction volume of 25 µl was made up with 20 µl mastermix containing all primers and probes and 50 copies of the plasmidIPC and 5 µl template solution.
All real-time PCR experiments were run with the following cycling parameters, 15 min at 95 °C followed by 45 cycles consisting of 15 s at 95 °C and 90 s at 60 °C.
The assay layout was as follows: four No Template Control (NTC) reactions with 5 µl 0.1× TE buffer instead of DNA template solution, however, containing 50 copies plasmidIPC and two positive control reactions (PosC) in duplicates each. All four PosC reactions comprised 50 copies of plasmid35S, plasmidNOS and plasmidFMV, while two reactions contained 50 copies of plasmidLY (PosC1), and the other two reactions contained 50 copies of plasmidSAMS (PosC2).
Evaluation criteria in relation to the respective positive control as reference; cycle threshold (Ct) cut-off and fluorescence intensity (dR) limit(Check)
Ct cut-off: mean Ct(PosCGMO/NTCIPC) +Ct
dR limit: mean dR(PosCGMO/NTCIPC)
dR limitCheck: mean dR(PosCFMV / LY/SAMS)
Test reaction scoring in GMO detection systems (35S, NOS, FMV, LY/SAMS resp.)
CtGMO sample ≤ CtGMO cut-off
dRGMO sample ≥ dRGMO limit
CtGMO sample ≤ CtGMO cut-off
dRGMO sample < dRGMO limit
CtFMV / LY/SAMS sample > CtFMV / LY/SAMS cut-off
dRFMV/LY/SAMS limitCheck ≤ dRFMV/LY/SAMS sample < dRFMV/LY/SAMS limit
CtGMO sample > CtGMO cut-off
dRGMO sample ≥ dRGMO limit
Ct35S/Nos sample > Ct35S/NOS cut-off
dR35S/Nos sample < dR35S/Nos limit
CtFMV / LY/SAMS sample > CtFMV / LY/SAMS cut-off
dRFMV/LY/SAMS sample < dRFMV/LY/SAMS limitCheck
Final result combining IPC and GMO screening results
GMO detection system
Selection of systems
35S: Cauliflower Mosaic Virus (CaMV) 35S promoter
NOS: Agrobacterium tumefaciens NOS terminator
FMV: Figwort Mosaic Virus (FMV) 34S promoter
SAMS: Transition from S-adenosyl-l-methionine synthetase (SAMS) promoter to Glycine max acetolactate synthase (gm-hra) gene
LY: Transition from Zea mayschloroplast transit peptide sequence for dihydrodipicolinate synthase to Corynebacterium glutamicum dihydrodipicolinate synthase (cordapA) gene encoding for a lysine-insensitive dihydropicolinate synthase enzyme
IPC: Sequence of non-plant origin
The detection systems for CaMV 35S promoter, NOS terminator and FMV 34S promoter were selected because these regulatory elements are most frequently present in GMOs. However, these elements do no longer provide complete screening coverage for maize and soybean due to new varieties on the market, which lack these elements. Therefore two new construct-specific PCR systems were developed and included in the hexaplex assay.
The integrated IPC system, detecting a sequence of non-plant origin, enabled the verification of absence of PCR inhibitors.
Hexaplex assay development
Properties of reporter dyes and non-fluorescent quenchers used for probe labelling
Excitation max. [nm]
Emission max. [nm]
Absorption max. [nm]
CAL Fluor 610
Overview of real-time PCR detection systems and the respective detection channels
Filter excitation max. [nm]
Filter emission max. [nm]
CAL Fluor 610 BHQ-2®
The primer and probe concentrations for all GMO detection systems were kept equal, while the IPC system was applied with lowered primer and probe concentrations.
Statistical indicators for uniform performance of the multiplexed real-time PCR systems
Target of Cy5 detection channel
rel. SD (dR)
rel. SD (dR)
Limit of detection absolute (LODabs)
The specificity testing performed in hexaplex assay format included DNA preparations from all commercially available GMs from soybean, maize, canola, cotton, rice, sugar beet, potato material and the respective non-GM plant DNAs as described above. All DNA preparations were tested in triplicates, and the obtained results for GM plant DNAs were in compliance with the expectations according to the theoretical presence of the tested GMO targets as recorded in publicly available GMO databases [23, 24]. Non-GM plant DNA preparations were tested negative in all GMO detection systems. To confirm specificity, amplicons of SAMS and LY PCR system were sequenced as well as amplicons of NOS and FMV PCR system, exemplarily.
Limit of detection absolute (LODabs)
Number of positive reactions out of 12 reactions each containing 0.693 copies of the respective positive control DNA tested in multiplex format
Results of validation experiments with spiked, (partially) inhibited DNA extracts from diverse sample matrices
Positive or inhibited
To check for matrix-specific effects, 44 sample materials from various matrices, which were 35S and NOS negative and showed no inhibition in pre-testing, were tested with the hexaplex screening assay. As a result, neither matrix-specific inhibition nor unspecific signals in any multiplexed PCR system were observed (data not shown).
Stability in assay performance after storage
dCt (mean CtVAR-mean CtREF)
3 days at RT
Several multiplex assays for GMO detection by conventional PCR have been described in the literature and succeeded for example in one case in a nonaplex PCR for detection and discrimination of nine GMO-related targets by agarose gel electrophoresis . In real-time PCR, the number of targets for multiplexing is mainly restricted to the real-time PCR platform constraints. To our knowledge, the highest multiplex level of a published real-time PCR assay for GMO detection based on TaqMan™ technology is a quadruplex real-time PCR assay . Therefore a major task in the development of a hexaplex assay was to overcome certain constraints of the real-time PCR platform. To achieve this, the real-time PCR platform needed to be specifically configured with a filter set to excite and detect ATTO425 dye-labelled probes, emitting in the blue spectrum. Furthermore the redundancy of a passive reference dye was demonstrated in homogeneity testing. All PCR systems of the hexaplex assay showed uniform performance without normalisation. The fluorophores for probe labelling (Table 4) for differential detection of the PCR products were chosen to match the filter sets of the real-time PCR platform (Table 5) while exhibiting a minimum overlap in the emission spectra. With this dye combination, all five detection channels of the real-time PCR platform were used, showing no significant crosstalk between the detection channels. For detection of a sixth real-time PCR system, one detection channel was chosen to be double-used.
The new generation of real-time PCR platforms extend the optical range in which dyes are excited and fluorescence is detected from UV to infrared wavelengths. This represents a significant advantage compared to laser based-real-time PCR platforms regarding the capability for multiplexing. In combination with newly developed dyes for oligonucleotide labelling, the multiplex level by means of real-time PCR has further potential to increase in the future.
The in-house validation data presented in this study shows that a hexaplex real-time PCR screening assay was developed, which proved to be fit for purpose for reliable detection of GM plants in food, feed and seed samples.
It was shown that multiplexing of real-time PCR systems is possible without a loss in sensitivity, presenting an assay with a limit of detection ≤ ten target copies in hexaplex format. There are critical issues in multiplex real-time PCR, which were taken into consideration in the development and successfully validated in this work.
One important aspect, which has been omitted in most validations on multiplex PCR assays for routine testing published to date, is competition between the multiplexed PCR systems. Especially for screening assays this is relevant, normally representing the first level of analysis in routine testing and thus dealing with various matrices and variable GMO contents. Such samples can have quite asymmetric target ratios. The validation of the hexaplex screening assay proved single-copy sensitivity for all GMO detection systems in the presence of 64 copies of all other GMO targets of each detection channel. Additionally the sensitivity and robustness of each GMO detection system was shown in a highly asymmetric target situation of ten target copies of one GMO target versus 1,000 copies of the GMO targets of each detection channel. This represents a broad working range for the analysis of food, feed and seed samples. To extend this range, single GMO detection systems were developed in parallel to the development of the hexaplex screening assay. All single GMO detection systems were duplexed with the IPC system used in the hexaplex screening assay and extensively validated for LODabs, IPC reliability, matrix effects and stability, referring to this study. Hence, negative results can be reconfirmed with the respective single GMO detection systems in case that a sample is beyond the validated working range regarding asymmetry of targets. To indicate the occurrence of non-validated asymmetric target situations, a feedback signal was incorporated in the developed evaluation spreadsheet tool. This consequently leads to a re-analysis of the negative results by the single GMO detection systems.
Another critical factor in multiplex PCR is the increased risk for artefacts through interactions between the oligonucleotides, which can lead to unspecific signals and even to false-positive results. In the validation process of the assay, this was tested under challenging conditions in stability testing, and as a result, no significant increase of the fluorescence signals was observed in NTC reactions. Additionally the absence of unspecific signals with DNA extracts of different sample matrices was demonstrated.
The IPC reliability validation showed the multiplexed IPC system being sensitive enough to indicate PCR inhibiting substances in the DNA preparation before each of the GMO detection systems was significantly suppressed. Integrated in the developed evaluation algorithm, this enabled the reliable and elegant verification of absence or presence of PCR inhibition. This procedure alleviated the need for separate controls like spiked reactions in parallel and is consequently a time- and cost-saving strategy.
As a summary, multiplex real-time PCR screening assays, as the newly developed hexaplex screening assay presented in this study, offer a sensitive and reliable detection platform in GMO analysis with several advantages: low risk of contamination, decreased turnaround time and high throughput testing with improved process costs.