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.
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.                                                                  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.
Laboratoire de Génétique, Biodiversité et Valorisation des Bioressources, Institut Supérieur de Biotechnologie de Monastir (ISBM), Université de Monastir, Avenue Tahar Haddad 5000, BP74, Monastir, Tunisia. maher.chaouachi@gmail.com
Maher
M
Chaouachi
Akram
A
Alaya
Imen Ben Haj
IB
Ali
Ahmed Ben
AB
Hafsa
Nesrine
N
Nabi
Aurélie
A
Bérard
Marcel
M
Romaniuk
Fethia
F
Skhiri
Khaled
K
Saïd
D027461:Beta vulgaris / Q000235:genetics*; D017931:DNA Primers / Q000378:metabolism; D018628:Gene Dosage / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics*; D014644:Genetic Variation; D030821:Plants, Genetically Modified; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results
D017931:DNA Primers