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

Analytical and Bioanalytical Chemistry, Mar 2018

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. Open image in new window Graphical abstract There 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. Keywords Digital PCR Droplet digital PCR Chip-based digital PCR Genetically modified organisms Quantification  Tigst Demeke and David Dobnik contributed equally to this work. Download article PDF Introduction Genetically modified organisms (GMOs) have already passed a two-decade milestone of presence on the market, and the number of GMOs as well as the worldwide area planted with biotech crops continues to increase steadily [1]. Many countries regulate the cultivation and trade of GMOs [2, 3], whereas others have at least some kind of authorization system and/or labelling requirements in place for GMOs present in food and feed chains [3, 4]. Labelling thresholds are relatively low in some countries; for example, in the European Union (EU) Regulation (EC) 1829/2003 [5] has set the labelling threshold for food products that contain, consist of or are produced from authorized GMOs at 0.9%. For feed samples there is an even lower threshold of 0.1% for GMOs, for which authorization is either pending or has expired (so-called low-level presence) [6]. Additionally, there is zero tolerance for unapproved GMOs in EU countries and some other countries. As a consequence, sensitive and accurate methods must be used for GMO detection and quantification to check product labelling compliance with the legislation. During the last 20 years, several approaches have been developed for GMO detection and quantification, and these are generally divided into two groups: protein-based and DNA-based methods. Because of several performance parameters, DNA-based methods are more widely accepted and used. One of the most promising DNA-based methods is digital polymerase chain reaction (dPCR), which is critically reviewed in this article. Overview of different technologies used for GMO detection Protein-based methods The most commonly used protein-based methods for detection and quantification of GMOs are enzyme-linked immunosorbent assay and use of lateral flow devices (LFDs; or lateral flow strips) [7]. Although protein-based methods are used less often than DNA-based methods, they have some advantages. For instance, LFDs are inexpensive, are simple to use, and provide quick detection of the presence or absence of proteins coded by the inserted GMO gene. They can be used on-site (e.g. at grain elevators) with minimum expertise and equipment requirements (e.g. in the format of a portable immunosensor as suggested for on-site GMO monitoring) [8]. LFDs are also available in a comb format for the detection of multiple GMOs. They are also useful for screening of plant seedlings for the presence of specific GMO traits, and can also be used for analysis of grain samples in which the proteins are not sheared. The quantitative aspect of protein-based methods is addressed with enzyme-linked immunosorbent assay, which provides quantitative test results using known reference standards and optical plate readers [9]. Protein-based methods have been used for verification of the identity preservation system for non-GMO grains (e.g. soybean). Nevertheless, protein-based methods also have some drawbacks. Development of antibodies is expensive and time-consuming, in some instances different levels of protein expression can occur, variation in protein content can occur in different tissues and cells, and perhaps most importantly, there is a lack of availability of protein-based methods for all GMO events [7, 10, 11]. All protein-based methods are specific only for the protein coded by the inserted GMO construct, and are not specific for the GMO trait. Proteins are also more sheared than DNA in processed samples (e.g. food products). Thus, protein-based methods are not suitable for identification and quantification in processed samples.

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

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms Tigst Demeke 0 1 2 David Dobnik 0 1 2 0 Department of Biotechnology and Systems Biology, National Institute of Biology , Večna pot 111, 1000 Ljubljana , Slovenia 1 Canadian Grain Commission, Grain Research Laboratory , 1404-303 Main Street, Winnipeg, MB R3C3G8 , Canada 2 David Dobnik 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. Digital PCR; Droplet digital PCR; Chip-based digital PCR; Genetically modified organisms; Quantification - Introduction Genetically modified organisms (GMOs) have already passed a two-decade milestone of presence on the market, and the number of GMOs as well as the worldwide area planted with biotech crops continues to increase steadily [ 1 ]. Many countries regulate the cultivation and trade of GMOs [ 2, 3 ], whereas others have at least some kind of authorization system and/ or labelling requirements in place for GMOs present in food and feed chains [ 3, 4 ]. Labelling thresholds are relatively low in some countries; for example, in the European Union (EU) Regulation (EC) 1829/2003 [ 5 ] has set the labelling threshold for food products that contain, consist of or are produced from authorized GMOs at 0.9%. For feed samples there is an even lower threshold of 0.1% for GMOs, for which authorization is either pending or has expired (so-called low-level presence) [ 6 ]. Tigst Demeke and David Dobnik contributed equally to this work. * Overview of different technologies used for GMO detection Protein-based methods The most commonly used protein-based methods for detection and quantification of GMOs are enzyme-linked immunosorbent assay and use of lateral flow devices (LFDs; or lateral flow strips) [ 7 ]. Although protein-based methods are used less often than DNA-based methods, they have some advantages. For instance, LFDs are inexpensive, are simple to use, and provide quick detection of the presence or absence of proteins coded by the inserted GMO gene. They can be used on-site (e.g. at grain elevators) with minimum expertise and equipment requirements (e.g. in the format of a portable immunosensor as suggested for on-site GMO monitoring) [ 8 ]. LFDs are also available in a comb format for the detection of multiple GMOs. They are also useful for screening of plant seedlings for the presence of specific GMO traits, and can also be used for analysis of grain samples in which the proteins are not sheared. The quantitative aspect of protein-based methods is addressed with enzyme-linked immunosorbent assay, which provides quantitative test results using known reference standards and optical plate readers [ 9 ]. Protein-based methods have been used for verification of the identity preservation system for non-GMO grains (e.g. soybean). Nevertheless, protein-based methods also have some drawbacks. Development of antibodies is expensive and time-consuming, in some instances different levels of protein expression can occur, variation in protein content can occur in different tissues and cells, and perhaps most importantly, there is a lack of availability of protein-based methods for all GMO events [ 7, 10, 11 ]. All protein-based methods are specific only for the protein coded by the inserted GMO construct, and are not specific for the GMO trait. Proteins are also more sheared than DNA in processed samples (e.g. food products). Thus, protein-based methods are not suitable for identification and quantification in processed samples. DNA-based methods Polymerase chain reaction (PCR) is the leading DNA-based method for GMO detection. A second generation of PCR— that is, real-time quantitative PCR (qPCR)—has been the gold standard for GMO trait detection and quantification for more than a decade. The PCR-based GMO detection analysis approach usually starts with qualitative screening for the presence of genetic elements c (...truncated)


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Tigst Demeke, David Dobnik. Critical assessment of digital PCR for the detection and quantification of genetically modified organisms, Analytical and Bioanalytical Chemistry, 2018, pp. 1-12, DOI: 10.1007/s00216-018-1010-1