NGS-based amplicon sequencing approach; towards a new era in GMO screening and detection

Arulandhu, Alfred J.; Dijk, Jeroen van; Staats, Martijn; Hagelaar, Rico; Voorhuijzen, Marleen M.; Molenaar, Bonnie; Hoof, Richard van; Li, Rong; Liu, Yang; Shi, Jiannong; Scholtens, I.M.J.; Kok, E.J.

doi:10.1016/j.foodcont.2018.06.014

Cited by 22 publications

(18 citation statements)

References 20 publications

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“…However, the use of next-generation sequencing technology's capacity to carry out massively parallel multiplexed quantitative amplicon sequencing provides a viable alternative, although it will require significant development effort. This approach has already been applied qualitatively in GMO screening [44]. Fields such as microbiological ecology have already applied this approach quantitatively [45].…”

Section: Discussionmentioning

confidence: 99%

A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant

Chhalliyil

Ilves

Kazakov

et al. 2020

Foods

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Discussion regarding the regulatory status of genome-edited crops has focused on precision of editing and on doubts regarding the feasibility of analytical monitoring compliant with existing GMO regulations. Effective detection methods are important, both for regulatory enforcement and traceability in case of biosafety, environmental or socio-economic impacts. Here, we approach the analysis question for the first time in the laboratory and report the successful development of a quantitative PCR detection method for the first commercialized genome-edited crop, a canola with a single base pair edit conferring herbicide tolerance. The method is highly sensitive and specific (quantification limit, 0.05%), compatible with the standards of practice, equipment and expertise typical in GMO laboratories, and readily integrable into their analytical workflows, including use of the matrix approach. The method, validated by an independent laboratory, meets all legal requirements for GMO analytical methods in jurisdictions such as the EU, is consistent with ISO17025 accreditation standards and has been placed in the public domain. Having developed a qPCR method for the most challenging class of genome edits, single-nucleotide variants, this research suggests that qPCR-based method development may be applicable to virtually any genome-edited organism. This advance resolves doubts regarding the feasibility of extending the regulatory approach currently employed for recombinant DNA-based GMOs to genome-edited organisms.

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Section: Discussionmentioning

confidence: 99%

A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant

Chhalliyil

Ilves

Kazakov

et al. 2020

Foods

View full text Add to dashboard Cite

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“…Features such as absolute quantification, avoidance of using standard curves, and high resilience to inhibitors, makes ddPCR a promising alternative for GM event detection (Rački et al, 2014;Corbisier et al, 2015). SGS technologies have also been proposed to comply with the requirements for GM traceability due to the ability to detect all target sequences in multiple samples without the development and validation of target-specific methods and reference material (Arulandhu et al, 2018). However, the requirement of bioinformatics knowledge for data analysis and more sophisticated devices limits its use in routine GM event detection (Park et al, 2017).…”

Section: Gm Traceabilitymentioning

confidence: 99%

“…In recent years, significant effort has been performed to replace the time-consuming and expensive qPCR screening procedure (Holst-Jensen et al, 2016;Salisu et al, 2017). As a result, other technologies are been evaluated including ddPCR (Dalmira et al, 2016;Dobnik et al, 2015;Köppel et al, 2015;Demeke et al, 2016;Dobnik et al, 2016;Gerdes et al, 2016;Głowacka et al, 2016;Iwobi et al, 2016;Grelewska-Nowotko et al, 2018;Niu et al, 2018;Corbisier and Emons, 2019;Giraldo et al, 2019), SGS (Willems et al, 2016;Fraiture et al, 2017;Arulandhu et al, 2018), DNA enrichment approaches (Arulandhu et al, 2016) and combined strategies of DNA walking and SGS (Fraiture et al, 2017).…”

Section: Gm Traceabilitymentioning

confidence: 99%

Safety Assessment of Genetically Modified Feed: Is There Any Difference From Food?

et al. 2019

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“…However, the protein-based methods depend on the expression level of the targeted proteins and a nondegraded structure and are not applicable if the genetic modification has no impact at the protein level [3]. To overcome these issues, a second approach, DNA-based methods, including polymerase chain reaction (PCR) [4], Southern blot [5], DNA microarrays [6], and next generation sequencing [7] technologies, has been developed. Among these techniques, quantitative PCR (qPCR) is the method of choice in the routine analysis of GMOs.…”

Section: Introductionmentioning

confidence: 99%

Transgenic Plant Detection Using an AuNPs Based SPR Biosensor

et al. 2019

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The intensive development and commercialization of genetically modified plants observed over the last decade has led to the development of transgenic detection methods that are rapid and sensitive. Among the strategies used for the detection/monitoring of genetically modified organisms (GMOs), surface plasmon resonance (SPR) meets the necessary criteria. This optical technique measures the changes in the refractive index in the vicinity of thin metal layers (i.e., gold) in response to biomolecular interactions occurring at a flat metal‒solution interface. Additionally, it allows the application of functionalized gold nanoparticles (AuNPs) in SPR research to enhance the signal intensity. In the present study, an SPR method, enhanced by the application of AuNPs, was developed to detect transgenic tobacco plants carrying a Streptococcus mutans antigen. The basis for the detection of the target DNA was the hybridization between the genomic DNA isolated from the leaves, stems, and roots of the transgenic tobacco and the biotinylated oligonucleotide probes immobilized onto a streptavidin (SA) sensor chip. SA-functionalized AuNPs coated with a second type of biotinylated probe were applied to increase the sensitivity of the detection method. Analysis of the results indicated that the constructed SPR-based sensor chip can potentially recognize complementary standard fragments (nonamplified genomic DNA) at concentrations as low as 1 pM. Thus, nonamplified transgenic DNA was detected using a label-free and real-time AuNPs-enhanced SPR biosensing method. This unique approach could be used to detect GMOs with high efficiency, even at a low detection limit, high repeatability, and with less time and a lower cost needed for each analysis.

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NGS-based amplicon sequencing approach; towards a new era in GMO screening and detection

Cited by 22 publications

References 20 publications

A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant

A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant

Safety Assessment of Genetically Modified Feed: Is There Any Difference From Food?

Transgenic Plant Detection Using an AuNPs Based SPR Biosensor

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