Rapid establishment of droplet digital PCR for quantitative GMO analysis

Köppel, René; Bücher, Thomas

doi:10.1007/s00217-015-2475-1

Cited by 42 publications

(32 citation statements)

References 14 publications

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“…We routinely used the same amount of primers as in the qPCR (12.5 pmol = 625 nM). In the study of Köppel and Bucher [5], increased primer concentrations up to 800 nM resulted in much better droplet separation that was similarly demonstrated by other studies [6]. Furthermore, for ddPCR a concentration of 900 nM is recommended (Droplet Digital ™ PCR Applications Guide).…”

Section: Resultsmentioning

confidence: 77%

“…If the rain is caused by biased amplification efficiency, another strategy to improve accuracy and avoid incorrect thresholds is adapting the assay to avoid or minimize the rain (and thus false positives and negatives samples). Such adaptations could be variations in the PCR program [5,6], primer concentrations [5,6], DNA digestion [15], DNA preparation [7] and others. Nevertheless, this does not necessarily mean that adaptation works for every assay and one single adjustment might not be enough.…”

Section: Introductionmentioning

confidence: 99%

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A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

et al. 2016

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The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as “rain” in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.

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Section: Resultsmentioning

confidence: 77%

Section: Introductionmentioning

confidence: 99%

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

et al. 2016

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“…The duplex digital droplet PCR developed and presented in this work was designed to quantify samples in the range of 0.02-2 ng/μl. The development strategy was published recently [14]. The test of the specificity revealed that no cross-talk took place between the MON87769 and the MON87705 systems although the primers and probes were the same as used in the AllSoyC.…”

Section: Resultsmentioning

confidence: 99%

“…Experiments to establish droplet digital PCR systems showed that a primer concentration of 0.4 μM in most cases successfully amplifies one single copy of the targeted DNA after 50 cycles, applying an annealing temperature of 60 °C [14]. This was the case for soybean lectin, MON87708, MON87769 and FG72.…”

Section: Design Of the Duplex Droplet Digital Pcr (Ddpcr)mentioning

confidence: 97%

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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“…The number of references to dPCR and its possible application in the field of GMO quantification have dramatically increased in recent years (Burns et al 2010;Corbisier et al 2010;Morisset et al 2013;Demeke T. et al 2014;Dobnik et al 2015;Koppel and Bucher 2015;Dobnik et al 2016;Félix-Urquídez et al 2015;Gerdes et al 2016;Zhu et al 2016). …”

Section: Introductionmentioning

confidence: 99%

In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

et al. 2017

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Common wheat is one of the most important staple food crops worldwide. However, unlike other important staple crops such as maize or soybean, genetically modified (GM) wheat is not yet present in the global food market. Nonetheless, in the recent past, the adventitious presence of GM glyphosatetolerant volunteers was reported in open wheat fields in the USA. The European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) was therefore called to develop a strategy to detect such unauthorised GM wheat in wheat samples by using both taxon-specific and screening tests. Two candidate common wheat taxon-specific real-time PCR methods were suggested, one targeting ssII-D gene coding for starch synthase and the other targeting waxy-D1 gene, coding for granule-bound starch synthase. In the present study, the two above-mentioned real-time PCR taxon-specific methods were in-house verified and compared, proposing droplet digital PCR (ddPCR) as a new tool for supporting the application of the European Network of GMO Laboratories (ENGL) established method performance criteria. Preliminary performance data of waxy-D1 and ssII-D methods in ddPCR format are shown too to give a contribution to the bridging process from the consolidated to the emerging quantitative PCR methodology.

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Rapid establishment of droplet digital PCR for quantitative GMO analysis

Cited by 42 publications

References 14 publications

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

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

In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

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