Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

doi:10.1038/srep35451

Cited by 108 publications

(83 citation statements)

References 26 publications

(36 reference statements)

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“…Recently, droplet digital PCR (ddPCR) emerged as a new technology that enables processing of ~20,000 up to 1,000,000 separate PCR reactions in a single tube [23, 24]. This method facilitates the accurate and precise quantitation of nucleic acid targets without the need for calibration curves and any external standards [23].…”

Section: Introductionmentioning

confidence: 99%

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

2017

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Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

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Section: Introductionmentioning

confidence: 99%

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

2017

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“…New multiplexing methods (e.g., multiplex ddPCR) do show some increased performance in comparison to simplex qPCR methods, as the number of targets detected in one analysis far exceeds the single target from simplex methods Dobnik et al 2015Dobnik et al , 2016Košir et al 2017). However, their main drawback is the investment in new equipment, additional personnel training costs, and the longer time for analysis of one sample.…”

Section: Discussionmentioning

confidence: 99%

Decision Support for the Comparative Evaluation and Selection of Analytical Methods: Detection of Genetically Modified Organisms as an Example

et al. 2018

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The selection of the best-fit-for-purpose analytical method to be implemented in the laboratory is difficult due to availability of multiple methods, targets, aims of detection, and different kinds and sources of more or less reliable information. Several factors, such as method performance, practicability, cost of setup, and running costs need to be considered together with personnel training when selecting the most appropriate method. The aim of our work was to prepare a flexible multicriteria decision analysis model suitable for evaluation and comparison of analytical methods used for the purpose of detecting and/or quantifying genetically modified organisms, and to use this model to evaluate a variety of changing analytical methods. Our study included selection of PCR-, isothermal-, protein-, microarray-, and next-generation sequencing-based methods in simplex and/or multiplex formats. We show that the overall result of their fitness for purpose is relatively similar; however, individual criteria or a group of related criteria exposed more substantial differences between the methods. The proposed model of this decision support system enables easy modifications and is thus suitable for any other application of complex analytical methods.

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“…The RainDrop system provides higher sensitivity (can detect very low concentrations), with millions of droplets generated per sample. The QX100/QX200 and RainDrop platforms are widely used for absolute quantification of GMOs [49, 53, 54, 56, 57, 69, 77]. The QX100 and QX200 systems create around 20,000 droplets per well and have a relatively high throughput (96-well plates are used) compared with the RainDrop platform, which allows analysis of only eight reactions at a time, which are in turn divided into millions of droplets.…”

Section: Digital Pcr Systems Used For Gmo Detectionmentioning

confidence: 99%

“…These modifications allow spatial separation of respective clusters of amplified targets on the basis of their fluorescence level. Two of the approaches (non-discriminating multiplexing and amplitude-based multiplexing) were reported for GMO quantification [53, 54, 56]. In the EU, Regulation (EC) No 1829/2003 specifies that quantification of the concentration of genetically modified material should be per ingredient (interpreted also as per species).…”

Section: Multiplex Quantification Of Gmo Events With Dpcrmentioning

confidence: 99%

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

Demeke¹,

Dobnik

2018

Anal Bioanal Chem

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110

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The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing. Graphical abstractThere are generally three different options for absolute quantification of genetically modified organisms (GMOs) using digital PCR: droplet- or chamber-based and droplets in chambers. All have in common the distribution of reaction mixture into several partitions, which are all subjected to PCR and scored at the end-point as positive or negative. Based on these results GMO content can be calculated.

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Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

Cited by 108 publications

References 26 publications

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

Decision Support for the Comparative Evaluation and Selection of Analytical Methods: Detection of Genetically Modified Organisms as an Example

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

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