Abstract
Key message
Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis.
Abstract
The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R 2 > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.
Similar content being viewed by others
Abbreviations
- C t :
-
Cycle threshold
- CTAB:
-
Cetyl trimethylammonium bromide
- dNTP:
-
Deoxynucleotide triphosphate
- EC:
-
European Commission
- EST:
-
Expressed sequence tag
- EU:
-
European Union
- EU-RL:
-
European Reference Laboratory
- GS:
-
Glutamine synthase
- HGC:
-
Haploid genome copy
References
AFNOR Standard XP-V-03-020-2 (2003) Détection et quantification des organismes végétaux génétiquement modifiés et produits dérivés. Partie 2: Méthodes basées sur la réaction de polymérisation en chaîne, pp 1–20. AFNOR (FR)
Anklam E, Gadani F, Heinze P, Pijnenburg H, Van den Eede G (2002) Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. Eur Food Res Technol 214:3–26
Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218
Barbau-Piednoir E, Lievens A, Vandermassen E, Mbongolo-Mbella EG, Leunda-Casi Nancy Roosens, Sneyers A, M Van Bulcke den M (2012) Four new SYBR_Green qPCR screening methods for the detection of Roundup Ready, LibertyLink and CryIAb traits in genetically modified products. Eur Food Res Technol 234:13–23
Bertheau Y, Diolez A, Kobilinsky A, Magin K (2002) Detection methods and performance criteria for genetically modified organisms. J AOAC Inter 85:801–808
Chaouachi M, Giancola S, Romaniuk M, Laval V, Bertheau Y, Brunel D (2007) A strategy for designing multi-taxa specific reference gene systems. Example of application—ppi phosphofructokinase (ppi-PPF) used for the detection and quantification of three taxa: maize (Zea mays), cotton (Gossypium hirsutum) and rice (Oryza sativa). J Agric Food Chem 55:8003–8010
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 Agric Food Chem 56:1818–1828
Codex Committee on Methods of Analysis and Sampling (2012) In-house method validation, Codex Alimentarius Commission, CX/MAS 98/8, Budapest, Hungary, 1998, November 23–27. http://www.fao.org/. Accessed April 2012
Community Reference Laboratory for GM Food and Feed (2012) Home. http://gmo-crl.jrc.ec.europa.eu/. Accessed April 2012
Dinon AZ, Prins TW, Van Dijk JP, Arisi AC, Scholtens MJ, Kok EJ (2011) Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms. Anal Bioanal Chem 400:1433–1442
Dohm JC, Lange C, Holtgräwe D, Rosleff Sörensen T, Borchardt D, Schulz B, Lehrach H, Weisshaar B, Himmelbauer H (2011) Palaeohexaploid ancestry for caryophyllales inferred from extensive gene-based physical and genetic mapping of the sugar beet genome (Beta vulgaris). Plant J. doi:10.1111/j.1365-313X.2011.04898.x
EN ISO 21571 (2002) International Organization for Standardization, Geneva, Switzerland
European Commission (2012) Community reference laboratory for GM food and feed. Event specific method for the quantification of sugar beet line H7-1 using real-time PCR (protocol, accessed April 2012)
Fagan J, Schoeler B, Haegert A, Moore J, Beeby J (2010) Performance assessment under field conditions of a rapid immunological test for transgenic. J. Int of Food Science and Technology 36:357–367
Ford-Lloyd BV, Williams JT (1975) A revision of Beta section Vulgares (Chenopodiacea), with a new light on the origin of cultivated beets. Bot J Lin Soc 71:89–1402
Gašparič MB, Tengs T, La Paz JL, Holst-Jensen A (2010) Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Anal Bioanal Chem 396:2023–2029
Holst-Jensen A, Bertheau Y, De Loose M, Grohmann L, Hamels S, Hougs L, Morisset D, Pecoraro S, Pla M, den Bulcke MV, Wulff D (2012) Detecting un-authorized genetically modified organisms (GMOs) and derived materials. Biotechnol Adv. doi:10.1016/j.biotechadv.2012.01.024
Kluga L, Folloni S, Van den Bulcke M, Van den Eede G, Querci M (2012) Applicability of the ‘‘Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection’’ in processed maize matrices. Eur Food Res Technol 234:109–118
Kodama T, Kuribara H, Minegishi Y, Futo S, Watai M, Sawada C, Watanabe T, Akiyama H, Maitani T, Teshima R, Furui S, Hino A (2009) Evaluation of modified PCR quantitation of genetically modified maize and soybean using reference molecules: interlaboratory study. J AOAC Int 92:223–233
Matsuoka T, Kawashima Y, Akiyama H, Miura HA (2000) Method of detecting recombinant DNAs from four lines of genetically modified maize. J Food Hyg Soc Jpn 41:137–143
Paternò A, Marchesi U, Gatto F, Verginelli D, Quarchioni C, Fusco C, Zepparoni A, Amaddeo D, Ciabatti I (2009) Finding the joker among the maize endogenous reference genes for genetically modified organism (GMO) detection. J Agric Food Chem 57:11086–11091
Sarachu M, Colet M (2005) wEMBOSS: a web interface for EMBOSS. Bioinformatics 21:540–541
Schneider K, Borchardt DC, Schäfer-Pregl R, Nagl N, Glass C, Jeppsson A, Gebhardt C, Salamini F (1999) PCR-Based cloning and segregation analysis of functional gene homologues in Beta vulgaris. Mol Gen Genet 262:515–524
Shindo Y, Kuribara H, Matsuoka T, Futo S, Sawada CA (2002) Validation of real-time PCR analysis for line-specific quantitation of genetically modified maize and soybean using new reference molecules. J AOAC Int 85:1119–1126
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17:1105–1109
Tranzschel W (1927) Die Arten der Gattung Beta. Bull Appl Bot Plant Breed 17:203–223
Trifa Y, Zhang D (2004) DNA content in embryo and endosperm of maize kernal (Zea mays L.): impact on GMO quantification. J Agric Food Chem 52:1044–1048
Wang C, Jiang L, Rao J, Liu Y, Yang LK, Zhang D (2010) Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR. J Agric Food Chem 58:11543–11547
Wang X, Teng D, Yang Y, Tian F, Guan Q, Wang J (2011) Construction of a reference plasmid molecule containing eight targets for the detection of genetically modified crops. Appl Microbiol Biotechnol 90:721–731
Acknowledgments
We thank all the INRA groups who provided us samples and all the members of the EPGV-INRA unit in Evry/France and MDO laboratory in INRA Versailles/France for their precious help for providing samples and for the paper redaction.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by J. Register.
Rights and permissions
About this article
Cite this article
Chaouachi, M., Alaya, A., Ali, I.B.H. et al. Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet “Beta vulgaris L.”: GMO application. Plant Cell Rep 32, 117–128 (2013). https://doi.org/10.1007/s00299-012-1346-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-012-1346-5