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Development of Real-time PCR Assays for the Detection of the pin II Terminator (tpinII) Used in GM Constructs and Its Donor Organism, Potato (Solanum tuberosum)

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Two singleplex TaqMan methods were developed for the detection of potato targets: one for the detection of the tpinII terminator, which is an emerging terminator used in GM constructs, and one for the detection of the endogenous StLS gene of potato. Performance criteria such as specificity and sensitivity were successfully tested for the two methods, taking into account the recommendations of international guidelines. The presence of the StLS target was checked in 16 potato cultivars. The StLS target is present at low copy number and can be used for quantitation purposes, as demonstrated on transgenic potatoes in this paper. The StLS target is an excellent candidate to replace the presently recommended endogenous target based on the UGP gene, which shows several disadvantages due to its high copy number and lack of specificity. The research also indicates that DNA can easily be extracted from different parts of potato tubers with a classical cetyltrimethylammonium bromide method.

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  • AFNOR XP V03-020-2 (2003) Détection et quantification des organismes végétaux génétiquement modifiés et produits dérivés : méthodes basées sur la réaction de polymérisation en chaîne. Norme expérimentale 2003

  • Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9(3):208–218

    Article  CAS  Google Scholar 

  • Bajji M, M’Hamdi M, Gastiny F, Delaplace P, Fauconnier ML, Du Jardin P (2007) Catalase inhibition alters suberization and wound healing in potato (Solanum tuberosum) tubers. Physiol Plant 129(3):472–483

    Article  CAS  Google Scholar 

  • Bennett MD, Leitch IJ (2005) Nuclear DNA amounts in angiosperms: progress, problems and prospects. Ann Botany 95(1):45–90

    Article  CAS  Google Scholar 

  • Block A, Debode F, Grohmann L, Hulin J, Taverniers I, Kluga L, Barbau-Piednoir E, Broeders S, Huber I, Van den Bulcke M, Heinze P, Berben G, Busch U, Roosens N, Janssen E, Žel J, Gruden K, Morisset D (2013) The GMOseek matrix : a decision support tool for optimizing the detection of genetically modified plants. BMC Bioinformatics 14(1):256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Broeders S, Huber I, Grohmann L, Berben G, Taverniers I, Mazzara M, Roosens N, Morisset D (2014) Guidelines for validation of qualitative real-time PCR methods. Trends Food Sci Technol 37(2):115–126

    Article  CAS  Google Scholar 

  • Chaouachi M, El Malki R, Berard A, Romaniuk M, Laval V, Brunel D, Bertheau Y (2008) Development of a real-time PCR method for the differential detection and quantification of four Solanaceae in GMO analysis : potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum). J Agricult Food Chem 56(6):1818–1828

    Article  CAS  Google Scholar 

  • Debode F, Marien A, Janssen E, Berben G (2010) Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects. Anal Bioanal Chem 396(6):2151–2164

    Article  CAS  PubMed  Google Scholar 

  • Debode F, Janssen E, Marien A, Berben G (2012) DNA detection by conventional and real-time PCR after extraction from vegetable oils. J Am Oil Chem 89(7):1249–1257

    CAS  Google Scholar 

  • Debode F, Janssen E, Berben G (2013) Development of 10 new screening PCR assays for GMO detection targeting promoters (pFMV, pNOS, pSSuAra, pTA29, pUbi, pRice actin) and terminators (t35S, tE9, tOCS, tg7). Eur Food Res Technol 236(4):659–669

    Article  CAS  Google Scholar 

  • Debode F, Marien A, Janssen E, Bragard C, Berben G (2017a) Influence of the amplicon length on real-time PCR results. Biotechnol Agron Soc Environ 21:3–11

    Google Scholar 

  • Debode F, Huber I, Macarthur R, Rischitor PE, Mazzara M, Herau V, Sebah D, Dobnik D, Broeders S, Roosens NH, Busch U, Berben G, Morisset D, Zel J (2017b) Inter-laboratory studies for the validation of two singleplex (tE9 and pea lectin) and one duplex (pat/bar) real-time PCR methods for GMO detection. Food Control 73:452–461

    Article  CAS  Google Scholar 

  • Doležel J, Greilhuber J (2010) Nuclear genome size: are we getting closer? Cytometry Part A 77(7):635–642

    Article  CAS  Google Scholar 

  • Dube S, Qin J, Ramakrishnan R (2008) Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS One 3(8):e2876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eckes P, Rosahl S, Schell J, Willmitzer L (1986) Isolation and characterization of a light-inducible, organ-specific gene from potato and analysis of its expression after tagging and transfer into tobacco and potato shoots. Mol Gen Genet 205(1):14–22

    Article  CAS  Google Scholar 

  • ENGL (2011) Verification of analytical methods for GMO testing when implementing interlaboratory validated methods. Guidance document EUR 24790 EN. last consultation on the 9 of Januari 2018

  • ENGL (2015) Definition of minimum performance requirement for analytical methods for GMO testing. CRL GM food and feed, JRC95544. Last consultation on the 18 of Augustus 2017

  • CCMAS (2010) Guidelines on performance criteria and validation of methods for detection, identification and quantification of specific DNA sequences and specific proteins in food. ALINORM 10/33/23, Appendix III. Last consultation on the 18 of Augustus 2017

  • ISO21571 (2005) Foodstuffs—methods of analysis for the detection of genetically modified organisms and derived products—nucleic acid extraction. ISO, Geneva

    Google Scholar 

  • Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N, Hirao T, Akiyama H, Goda Y, Toyada M, Hino A (2002) Novel reference molecules for quantitation of genetically modified maize and soybean. J AOAC Int 85(5):1077–1089

    CAS  PubMed  Google Scholar 

  • Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J (2000) 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 28:655–661

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • M’Hamdi M, Bettaieb T, Harbaoui Y, Mougou AA, du Jardin P (2009) Insight into the role of catalases in salt stress in potato (Solanum tuberosum L.) Biotechnol Agron Soc Envir 13(3):373–379

    Google Scholar 

  • Praca-Fontes MM, Carvalho CR, Clarindo WR, Cruz CD (2011) Revisiting the DNA C-values of the genome size-standards used in plant flow cytometry to choose the “best primary standards”. Plant Cell Rep 30:1183–1191

    Article  CAS  PubMed  Google Scholar 

  • Randhawa GJ, Singh M, Sharma R (2009) Validation of ST-LS1 as an endogenous reference gene for detection of AmA1 and cry1Ab genes in genetically modified potatoes using multiplex and real time PCR. Am J Pot Res 86(5):398–405

    Article  CAS  Google Scholar 

  • Sambrook J, Rusell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Savini C, Foti N, Mazzara M, Charles-Delobel C, Van Den Eede G (2006) Event-specific method for the quantification of event EH92-527-1 potato using real-time PCR—validation report and protocol—sampling and DNA extraction of potato. EUR 22358 EN, JRC34758, Method for DNA extraction from freeze and dried potato tubers. Joint Research-European Commission Biotechnology GMOs Unit, Varese.

  • Stockhaus J, Eckes P, Blau A, Schell J, Willmitzer L (1987) Organ-specific and dosage-dependent expression of the leaf/stem specific gene from potato after tagging and transfer into potato and tobacco plants. Nucleic Acids Res 15:3479–3491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stupar RM, Beaubien KA, Jin W, Song J, Lee MK, Wu C, Zhang HB, Han B, Jiang J (2006) Structural diversity and differential transcription of the patatin multicopy gene family during potato tuber development. Genetics 172(2):1263–1275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Taverniers I, Van Bockstaele E, De Loose M (2004) Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods. Anal Bioanal Chem 378(5):1198–1207

    Article  CAS  PubMed  Google Scholar 

  • Taverniers I, Papazova N, Allnutt T, Baumler S, Bertheau Y, Esteve T, Freyer R, Gruden K, Kuznetzov B, Luis La Paz J, Nadal A, Pla M, Vojvoda D, Wulff D, Zhang D (2012) Harmonised reference genes and PCR assays for GMO quantification. GM and Non-GM food supply chains: co-existence and traceability In: Bertheau Y (ed). Wiley-Blackwell, Oxford, pp 273–292.

  • Wolf C, Hübner P, Lüthy J, Pauli U (2001) Detection of potato DNA from potato tubers and processed food products by means of PCR. Mitt Lebensmittelunters Hyg 92(2):159–167

    CAS  Google Scholar 

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We are very grateful to Frédéric Gastiny of the University of Liège (Gembloux Agro-Bio Tech, Belgium) for providing the GM potato lines and Yordan Muhovski of CRA-W for his kind help with Southern blots.


The StLS work was carried out thanks to a grant from the Belgian Federal Science Policy Office. The development of the tpinII was funded by the Belgian Federal Public Service for Health, Food Chain Safety and Environment through the contract UGMMONITOR (convention RF 11/6242).

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Correspondence to Frederic Debode.

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Frederic Debode declares that he has no conflict of interest. Kamila Zdeňková declares that she has no conflict of interest. Eric Janssen declares that he has no conflict of interest. Anette Tizolova declares that she has no conflict of interest. Patrick du Jardin declares that he has no conflict of interest. Gilbert Berben declares that he has no conflict of interest. Kateřina Demnerova declares that she has no conflict of interest.

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This article does not contain studies with human or animal subjects. Samples used are food samples.

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Debode, F., Zdeňková, K., Janssen, E. et al. Development of Real-time PCR Assays for the Detection of the pin II Terminator (tpinII) Used in GM Constructs and Its Donor Organism, Potato (Solanum tuberosum). Food Anal. Methods 11, 2172–2180 (2018).

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