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

AbstractThe 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.

[1]  Hugo Germain,et al.  Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data , 2017, Scientific Reports.

[2]  Christopher M. Hindson,et al.  Absolute quantification by droplet digital PCR versus analog real-time PCR , 2013, Nature Methods.

[3]  H. Doi,et al.  Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species. , 2015, Environmental science & technology.

[4]  Dabing Zhang,et al.  Visual detection of multiple genetically modified organisms in a capillary array. , 2017, Lab on a chip.

[5]  S H Neoh,et al.  Quantitation of targets for PCR by use of limiting dilution. , 1992, BioTechniques.

[6]  Arne Holst-Jensen,et al.  Testing for genetically modified organisms (GMOs): Past, present and future perspectives. , 2009, Biotechnology advances.

[7]  Marie-Alice Fraiture,et al.  How Can We Better Detect Unauthorized GMOs in Food and Feed Chains? , 2017, Trends in biotechnology.

[8]  J. Griffith,et al.  Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment. , 2015, Water research.

[9]  Philippe Corbisier,et al.  Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number , 2009, Analytical and bioanalytical chemistry.

[10]  D. J. Perry,et al.  Adventitious presence of GMOs: Scientific overview for Canadian grains , 2006 .

[11]  Jana Žel,et al.  ALF: a strategy for identification of unauthorized GMOs in complex mixtures by a GW-NGS method and dedicated bioinformatics analysis , 2017, Scientific Reports.

[12]  Mojca Milavec,et al.  Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR , 2013, PloS one.

[13]  Olivier Thas,et al.  Quality control of digital PCR assays and platforms , 2017, Analytical and Bioanalytical Chemistry.

[14]  Arne Holst-Jensen,et al.  Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products , 2016, Analytical and Bioanalytical Chemistry.

[15]  Dany Morisset,et al.  Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection , 2016, Scientific Reports.

[16]  Hans-Ulrich Waiblinger,et al.  A practical approach to screen for authorised and unauthorised genetically modified plants , 2010, Analytical and bioanalytical chemistry.

[17]  Guillaume P. Gruère,et al.  A Review of International Labeling Policies of Genetically Modified Food to Evaluate India's Proposed Rule , 2007 .

[18]  J. Stave.  Protein immunoassay methods for detection of biotech crops: applications, limitations, and practical considerations. , 2002, Journal of AOAC International.

[19]  A. Iwobi,et al.  Droplet digital PCR for routine analysis of genetically modified foods (GMO) – A comparison with real-time quantitative PCR , 2016 .

[20]  Jim F Huggett,et al.  Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus. , 2014, Analytical chemistry.

[21]  P. Laurent-Puig,et al.  Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. , 2013, Clinical chemistry.

[22]  Assessment of droplet digital PCR for absolute quantification of genetically engineered OXY235 canola and DP305423 soybean samples , 2014 .

[23]  C. Foy,et al.  The applicability of digital PCR for the assessment of detection limits in GMO analysis , 2010 .

[24]  Kristina Gruden,et al.  GMO quantification: valuable experience and insights for the future , 2014, Analytical and Bioanalytical Chemistry.

[25]  Esther J. Kok,et al.  DNA enrichment approaches to identify unauthorized genetically modified organisms (GMOs) , 2016, Analytical and Bioanalytical Chemistry.

[26]  Esther J. Kok,et al.  Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing , 2014, Analytical and Bioanalytical Chemistry.

[27]  Maja Ravnikar,et al.  Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples , 2014, Plant Methods.

[28]  F. Gatto,et al.  Development and applicability of a ready-to-use PCR system for GMO screening. , 2016, Food chemistry.

[29]  Application of Digital PCR in the Analysis of Transgenic Soybean Plants , 2016 .

[30]  Benjamin J. Hindson,et al.  Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification , 2011, Analytical chemistry.

[31]  Rainer Fischer,et al.  Characteristics of Genome Editing Mutations in Cereal Crops. , 2017, Trends in plant science.

[32]  Il-Hoon Cho,et al.  Development of a chemiluminometric immunosensor array for on-site monitoring of genetically modified organisms , 2011 .

[33]  J. Xing,et al.  Absolute quantification of genetically engineered traits with droplet digital PCR: Effect of DNA treatments and spiking with non-target DNA , 2016 .

[34]  A. Olper,et al.  International trade and endogenous standards: the case of GMO regulations , 2012, World Trade Review.

[35]  Alexandra Bogožalec Košir,et al.  Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines , 2017, Scientific Reports.

[36]  J. Emerson,et al.  Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection , 2019, BMC Biotechnology.

[37]  H. Waiblinger,et al.  Validation and collaborative study of a P35S and T-nos duplex real-time PCR screening method to detect genetically modified organisms in food products , 2008 .

[38]  Olivier Thas,et al.  ddpcRquant: threshold determination for single channel droplet digital PCR experiments , 2015, Analytical and Bioanalytical Chemistry.

[39]  Jim F Huggett,et al.  Evaluation of digital PCR for absolute DNA quantification. , 2011, Analytical chemistry.

[40]  Sofie Rutsaert,et al.  Digital PCR as a tool to measure HIV persistence , 2018, Retrovirology.

[41]  K. Gruden,et al.  Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot , 2014, Analytical and Bioanalytical Chemistry.

[42]  Monika Singh,et al.  Real-time and visual loop-mediated isothermal amplification: Efficient GMO screening targeting pat and pmi marker genes , 2017 .

[43]  D. Deforce,et al.  An integrated strategy combining DNA walking and NGS to detect GMOs. , 2017, Food chemistry.

[44]  Maja Ravnikar,et al.  Droplet digital PCR for absolute quantification of pathogens. , 2015, Methods in molecular biology.

[45]  Youping Yin,et al.  Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri , 2016, PloS one.

[46]  M. Baker.  Digital PCR hits its stride , 2012, Nature Methods.

[47]  J. Madic,et al.  Three-color crystal digital PCR , 2016, Biomolecular detection and quantification.

[48]  Ulrich Busch,et al.  Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms , 2016, Biomolecular detection and quantification.

[49]  J. Stave,et al.  Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients , 2000 .

[50]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[51]  Alison S. Devonshire,et al.  International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant. , 2017, Analytical chemistry.

[52]  Hai-Feng Ji,et al.  Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification method. , 2014, Food chemistry.

[53]  J. Zel,et al.  Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops. , 2013, Journal of agricultural and food chemistry.

[54]  Arne Holst-Jensen,et al.  Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction. , 2015, Analytical chemistry.

[55]  K. Kinzler,et al.  Digital PCR. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[56]  G. Berben,et al.  Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed. , 2013, Journal of agricultural and food chemistry.

[57]  A Lievens,et al.  Measuring Digital PCR Quality: Performance Parameters and Their Optimization , 2016, PloS one.

[58]  S. Lucas,et al.  Assessment of a direct hybridization microarray strategy for comprehensive monitoring of genetically modified organisms (GMOs). , 2016, Food chemistry.

[59]  A. Holck,et al.  Quantitative, multiplex ligation-dependent probe amplification for the determination of eight genetically modified maize events , 2009 .

[60]  N. L. Innes.  Global Status of Commercialized Biotech/GM Crops: 2005. ISAAA Briefs No. 34. By C. James. Ithaca, NY, USA: ISAAA (2005), pp. 46, US$50.00. ISBN 1-892456-38-9 , 2006, Experimental Agriculture.

[61]  W. Cho,et al.  Applications of digital PCR in precision medicine , 2017 .

[62]  Mojca Milavec,et al.  Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA , 2017, Analytical and Bioanalytical Chemistry.

[63]  Huiyu Low,et al.  Clarity™ digital PCR system: a novel platform for absolute quantification of nucleic acids , 2017, Analytical and Bioanalytical Chemistry.

[64]  Jim F Huggett,et al.  Considerations for digital PCR as an accurate molecular diagnostic tool. , 2015, Clinical chemistry.

[65]  Hendrik Emons,et al.  DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials , 2015, Analytical and Bioanalytical Chemistry.

[66]  Roberto Bellotti,et al.  Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR , 2017, Analytical and Bioanalytical Chemistry.

[67]  Jim F Huggett,et al.  RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia. , 2016, Clinical chemistry.

[68]  L. Hougs,et al.  Detecting un-authorized genetically modified organisms (GMOs) and derived materials. , 2012, Biotechnology advances.

[69]  J. Wong,et al.  Advantages of using the QIAshredder instead of restriction digestion to prepare DNA for droplet digital PCR. , 2014, BioTechniques.

[70]  Alexandra S. Whale,et al.  Fundamentals of multiplexing with digital PCR , 2016, Biomolecular detection and quantification.

[71]  D. Deforce,et al.  Current and New Approaches in GMO Detection: Challenges and Solutions , 2015, BioMed research international.

[72]  Shuifang Zhu,et al.  A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment , 2015, Scientific Reports.

[73]  P. Corbisier,et al.  Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction , 2010, Analytical and bioanalytical chemistry.

[74]  G Ronald Jenkins,et al.  Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits , 2010, Analytical and bioanalytical chemistry.

[75]  J. Kramar,et al.  Method for Measuring the Volume of Nominally 100 um Diameter Spherical Water-in-Oil Emulsion Droplets , 2016 .

[76]  J. Mano,et al.  Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize. , 2011, Shokuhin eiseigaku zasshi. Journal of the Food Hygienic Society of Japan.

[77]  Jing Wang,et al.  Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material , 2015, Scientific Reports.

[78]  Alexandra S. Whale,et al.  Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples , 2013, PloS one.