Aliki Xanthopoulou1, Ioannis Ganopoulos2, Paschalitsa Tryfinopoulou3, Efstathios Z Panagou3, Maslin Osanthanunkul4,5, Panagiotis Madesis1, Dimosthenis Kizis6. 1. Institute of Applied Biosciences, CERTH, Thessaloniki, Greece. 2. Institute of Plant Breeding and Genetic Resources ELGO-DEMETER, Thessaloniki, Greece. 3. Laboratory of Food Microbiology and Biotechnology, Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, Greece. 4. Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand. 5. Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Chiang Mai, Thailand. 6. Laboratory of Mycology, Department of Phytopathology, Benaki Phytopathological Institute, Athens, Greece.
Abstract
BACKGROUND: Aspergillus is a diverse genus of fungi with high economic and social impact. Various species that belong to section Nigri (black aspergilli) are common agents of grape spoilage and potent producers of ochratoxin A (OTA), a mycotoxin associated with various nephrotoxic and immunotoxic effects in humans. Black aspergilli are difficult to classify following only phenotypic criteria; thus chemotaxonomic and molecular methods are employed in parallel with phenotypic ones for species characterization. These approaches, though accurate and replicable, require more than one individual step and are to a certain extent laborious when a rapid identification of these species is required. RESULTS: The aim of this study was to develop a high-resolution melting polymerase chain reaction (HRM-PCR) assay as a rapid method for identification of Aspergillus spp. section Nigri isolates and their detection in grape samples. Melt curve analysis of amplicons originating from the internal transcribed spacer 2 (ITS2) ribosomal region generated species-specific HRM curve profiles, enabling the accurate differentiation of the analyzed genotypes. Furthermore, the assay was able to identify A. carbonarius, A. tubingensis, A. niger, A. ibericus and A. japonicus in grape samples artificially inoculated with conidia of these fungi. CONCLUSION: To our knowledge this is the first report on the development of an HRM-PCR assay for the identification of black Aspergillus species in grape samples.
BACKGROUND:Aspergillus is a diverse genus of fungi with high economic and social impact. Various species that belong to section Nigri (black aspergilli) are common agents of grape spoilage and potent producers of ochratoxin A (OTA), a mycotoxin associated with various nephrotoxic and immunotoxic effects in humans. Black aspergilli are difficult to classify following only phenotypic criteria; thus chemotaxonomic and molecular methods are employed in parallel with phenotypic ones for species characterization. These approaches, though accurate and replicable, require more than one individual step and are to a certain extent laborious when a rapid identification of these species is required. RESULTS: The aim of this study was to develop a high-resolution melting polymerase chain reaction (HRM-PCR) assay as a rapid method for identification of Aspergillus spp. section Nigri isolates and their detection in grape samples. Melt curve analysis of amplicons originating from the internal transcribed spacer 2 (ITS2) ribosomal region generated species-specific HRM curve profiles, enabling the accurate differentiation of the analyzed genotypes. Furthermore, the assay was able to identify A. carbonarius, A. tubingensis, A. niger, A. ibericus and A. japonicus in grape samples artificially inoculated with conidia of these fungi. CONCLUSION: To our knowledge this is the first report on the development of an HRM-PCR assay for the identification of black Aspergillus species in grape samples.