title: Real-Time PCR Assay Targeting the veA Gene for Quantification of Aspergillus carbonarius in Grapes.
abstract: In this work, a SYBR Green I real-time PCR method has been developed for the detection and quantification of Aspergillus carbonarius in grapes by targeting the veA gene with a primer pair (veAF4/veAR4) that specifically amplifies a 91-bp PCR product. The quantification of the fungal DNA was performed by generation of standard curves for two A. carbonarius strains, using spectrophotometrically measured DNA quantities (Log) with a linearity range from 50 to 5 × 10(-4) ng of DNA. A high positive correlation (R(2) > 0.99) between exponential increases of DNA and real-time PCR threshold cycles showed a high amplification efficiency for the assay (E values 100.06 and 101.51%, respectively). Quantification of the fungal genomic DNA in grape samples artificially inoculated with A. carbonarius conidia was successfully performed with a minimum threshold of 10(4) conidia per g of grape berry. The assay developed would allow reliable, specific, and efficient detection and quantification of A. carbonarius in grapes.
authors.affiliation: Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece; Laboratory of Mycology, Department of Phytopathology, Benaki Phytopathological Institute, St. Delta 8, 14561, Athens, Greece. dimosthenis_kizis@yahoo.gr.
authors.forename: Dimosthenis
authors.initials: D
authors.lastname: Kizis
authors.affiliation: Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece.
authors.forename: George-John E
authors.initials: GJ
authors.lastname: Nychas
authors.affiliation: Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece.
authors.forename: Efstathios Z
authors.initials: EZ
authors.lastname: Panagou
mesh_terms: D001230:Aspergillus / Q000235:genetics*; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D005638:Fruit / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013172:Spores, Fungal; D027843:Vitis / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal