Multiplex real-time PCR for the detection and quantification of DNA from four transgenic soy Mon89788, A5547-127, Roundup Ready, A2704-12 and lectin

Köppel, René; Velsen, Franziska van; Felderer, Nora; Bücher, Thomas

doi:10.1007/s00217-012-1730-y

Cited by 11 publications

(11 citation statements)

References 16 publications

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developed with reasonable input [1][2][3][4][5]. A drawback of these analytical methods is the required reference material with known amount of GMO to calibrate the result of the unknown sample.

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mentioning

confidence: 99%

Droplet digital PCR versus multiplex real-time PCR method for the detection and quantification of DNA from the four transgenic soy traits MON87769, MON87708, MON87705 and FG72, and lectin

Köppel¹,

Bücher²,

Frei³

et al. 2015

Eur Food Res Technol

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developed with reasonable input [1][2][3][4][5]. A drawback of these analytical methods is the required reference material with known amount of GMO to calibrate the result of the unknown sample. Reference material itself introduces variation due to factors influencing the variation using PCR methods (e.g. inhibition, inherent measurement uncertainty). In addition, when analysing small series of samples, the required standards make a substantial amount of the reactions. Digital PCR may solve this inefficiency and inherent measurement uncertainty by making the use of reference material dispensable [6-9]. This saves not only expenses and work, but in cases where no reference material is available, it remains the only way to quantify at all.As the first step of the detection of gm plants, screening methods detecting target sequences present in many different gm plants are commonly used [10,11]. However, due to the lack of such sequences, in many cases screening methods developed so far do not detect gm soy events. For the detection of these soy events, single event-specific methods have to be used for each gm plant. Multiplex real-time PCR for the simultaneous detection of different gm soy can circumvent this time-and cost-consuming procedure. In addition, quantitative results of gm amount can be obtained. Therefore, we developed first a multiplex real-time PCR system for the quantitative determination of MON87769, MON87708, MON87705 and FG72 simultaneously (called AllSoyC). Additionally, we developed 4 duplex droplet digital PCR systems for the determination of the lectin (housekeeping gene) and the appropriate transgene marker gene. The results of both methods were compared [12] to assess whether quantification without using reference material is feasible and which method is more promising for the quantification of unknown samples.Abstract New genetically modified (gm) soy crops are going to be released for human consumption. Therefore, analytical skills have to be developed towards an efficient detection and determination of GMO content in feed and food. Existing approaches to screen gm plants do not detect new gm soy traits in many cases. Therefore, a multiplex quantitative real-time PCR system was developed and characterized for the four new transgenic soy traits MON87769, MON87708, MON87705 and FG72 to avoid time-and cost-consuming application of single event detection. It showed amplification efficiency, correlation and limit of quantification similar to the single PCR systems applied. The droplet digital PCR showed increased specificity. In parallel, we developed four duplex droplet digital PCR systems and compared the results from both methods. This showed that both approaches are fit for routine diagnostics. Real-time PCR may be more suited for screening as it is very cost-efficient. Digital PCR may be more suitable for quantitative analysis as it exhibited a measurement uncertainty of only 17 % or below for a single reaction.

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“…

developed with reasonable input [1][2][3][4][5]. A drawback of these analytical methods is the required reference material with known amount of GMO to calibrate the result of the unknown sample.

…”

mentioning

confidence: 99%

Droplet digital PCR versus multiplex real-time PCR method for the detection and quantification of DNA from the four transgenic soy traits MON87769, MON87708, MON87705 and FG72, and lectin

Köppel¹,

Bücher²,

Frei³

et al. 2015

Eur Food Res Technol

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“…The amplification efficiencies and correlations do not fulfil the ENGL minimum performance requirements for GMO quantification methods [15]. Nevertheless, the measurement uncertainties seem not to be impaired significantly.…”

Section: Correlation Of Performance and Measurement Uncertainty And Zmentioning

confidence: 97%

“…15 ng DNA per assay depending on the reference material and its % gm). The DNA was isolated from reference material.…”

mentioning

confidence: 99%

“…But this system works only as single-analyte system [12]. Since multiplex real-time PCR systems have shown to be feasible and can be developed with reasonable effort and time [13][14][15][16], the efficiency of PCR analysis increased significantly delivering also quantitative data.We developed in this work five multiplex real-time PCR systems for the detection and quantification of 20 different gm traits. We validated these systems considering international guidelines [17][18][19][20][21][22].…”

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confidence: 99%

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Five pentaplex real-time PCR systems for the efficient determination of 20 genetically modified maize traits in food

Köppel¹,

Ganeshan²,

Velsen³

et al. 2016

Eur Food Res Technol

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gm traits from samples without gm traits. Samples showing gm Maize contents may contain a mixture of different gm Maize traits. Such samples therefore require extensive analytical investigations using many PCR reactions and high amount of template DNA. This is time consuming and expensive. To make this analysis more efficient, multiplex PCR systems were developed. The first ones used detection by gel electrophoresis [1][2][3][4][5]. These systems were based on long amplicons making them distinguishable after gel electrophoresis; the results were qualitative only. Long amplicons often cannot be amplified as the template DNA is degraded during processing of the food product. This is a severe disadvantage as it makes detection of GMO often impossible. Capillary gel electrophoresis can distinguish smaller amplicons. Such systems were developed as well [6][7][8][9][10]. A further innovation was made when using ligation dependent amplification allowing the amplification of very short amplicons and as a consequence enabling the analysis of highly degraded products [11]. However, these techniques require a capillary electrophoresis equipment. To reduce costs of equipment, loop-mediated isothermal amplification systems were also developed. But this system works only as single-analyte system [12]. Since multiplex real-time PCR systems have shown to be feasible and can be developed with reasonable effort and time [13][14][15][16], the efficiency of PCR analysis increased significantly delivering also quantitative data.We developed in this work five multiplex real-time PCR systems for the detection and quantification of 20 different gm traits. We validated these systems considering international guidelines [17][18][19][20][21][22]. The correlation of amplification performance factors and measurement uncertainty (MU) was investigated. To assess accuracy, proficiency tests were performed. The influence of the difference matrices were assayed by analysing samples from daily routine.Abstract According to the EU and Swiss food legislation, only authorised traits of transgene plants are allowed to be imported and sold to the consumer. In order to control imports of maize products from retailers, efficient and reliable methods for the detection and quantification are a prerequisite. Actually, in the EU, more than 25 gm Maize traits were authorised and are allowed to be sold. To ensure correct labelling and exclude unauthorised gm traits from sale, five multiplex real-time polymerase chain reaction systems were developed and validated. These 20 systems for the most prevalent maize gm traits allow relative multiplex quantification and/or delta-delta Ct method quantification in routine in a reliable, cost-and time-efficient way.

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“…These multiplex TaqMan PCR assays are based on probes labelled by up to five different fluorescent dyes for simultaneous detection of the different target sequences. Another type of multiplex assay is the combination of event-specific real-time PCR methods that allows the detection and relative quantification of several GM soybean lines (Köppel et al 2012(Köppel et al , 2014. As a prerequisite for application, these assays require a real-time PCR instrument that can efficiently discriminate between the different fluorescent dyes without spectral overlap and crosstalk.…”

Section: Introductionmentioning

confidence: 99%

Screening for six genetically modified soybean lines by an event-specific multiplex PCR method: Collaborative trial validation of a novel approach for GMO detection

Grohmann

Belter

Speck

et al. 2016

J Consum Prot Food Saf

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This study presents a novel approach to detect genetically modified (GM) plant events that are not covered by common GMO screening methods. It is based on a simplified multiplex assay which merges the event-specific real-time PCR methods for the detection of six GM soybean lines (MON 87701, MON 87708, MON 87769, DP-305423, CV-127 and DAS-68416). The use of two different fluorescent dyes facilitates the subsequent analysis for identification of the GM event. The multiplex PCR method was validated in a collaborative study trial with 16 participating laboratories. Each laboratory received eight samples containing low levels (0.1% or 0.03% m/m) of one or two GM soybean lines and four GMnegative samples. Data of 720 PCR analyses were evaluated and a false-positive rate of 0.3% and a falsenegative rate of 3.9% was observed, respectively. The limits of detection (LOD 95%) were calculated based on modelling the probability of detection (POD) and show satisfactory sensitivity and reproducibility for the assay. Furthermore, we discuss the modularity and applicability of event-specific multiplex PCR systems for the detection of GM events that are not covered by screenings.

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Multiplex real-time PCR for the detection and quantification of DNA from four transgenic soy Mon89788, A5547-127, Roundup Ready, A2704-12 and lectin

Cited by 11 publications

References 16 publications

Droplet digital PCR versus multiplex real-time PCR method for the detection and quantification of DNA from the four transgenic soy traits MON87769, MON87708, MON87705 and FG72, and lectin

Droplet digital PCR versus multiplex real-time PCR method for the detection and quantification of DNA from the four transgenic soy traits MON87769, MON87708, MON87705 and FG72, and lectin

Five pentaplex real-time PCR systems for the efficient determination of 20 genetically modified maize traits in food

Screening for six genetically modified soybean lines by an event-specific multiplex PCR method: Collaborative trial validation of a novel approach for GMO detection

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