A molecular early detection method of Aspergillus carbonarius on grape and a novel analysis for Ochratoxin A in wine

Patrizia De Rossi1, Massimo Reverberi2, Alessandra Ricelli3, Domenico Caputo2, Giampiero De Cesare2, Augusto Nascetti2, Riccardo Scipinotti2, Corrado Fanelli2

Author address: 

1ENEA CR Casaccia, Roma, Italy, 2Universití  La Sapienza, Roma, Italy, 3CNR, Roma, Italy

Abstract: 

Aspergillus carbonarius is an important ochratoxin A (OTA) producing fungus which is responsible for toxin contamination of grapes and wine. OTA is a secondary metabolite produced by fungi belonging to Aspergillus and Penicillium genera which has been shown to be nephrotoxic, nephrocarcinogenic, teratogenic and immunosuppressive. The objective of this research was to investigate the presence of A. carbonarius in grapes and of OTA in wine. Two PCR-based assays have been developed to detect the presence of A. carbonarius in grapes by designing specie-specific primers on the basis of Internal Transcribe Spacers of rDNA units (Ac-ITS) and of the polyketide synthases (Ac-PKS) sequences. These specific primers were used for A. carbonarius detection in grapes. For early detection of Ochratoxin A the performance of a system based on hydrogenated amorphous silicon photosensors was investigated. This device is based on the measurement of the photocurrent induced in a hydrogenated amorphous silicon (a-Si:H) photodiode by the fluorescence of the mycotoxin excited by a UV radiation. The results of PCR using DNA extracted from various grape-contaminating fungi show that only A. carbonarius DNA was amplified with Ac-ITS and Ac-PKS primers. None of the other species gave a positive result with this PCR primers set used. Concerning OTA detection in wine the photocurrent measured by the sensor is due to the mycotoxin and the value is proportional to its quantity. PCR-based methods that target DNA are considered a good alternative with respect traditional diagnostic methods for early detection because of their high specificity and sensitivity. This PCR analysis was also successfully employed to detect A. carbonarius in grape. Currently, the most of the methods available for the determination of OTA in wine are based on an extraction step, a clean-up passage and determination by High Performance Liquid Chromatografy (HPLC) with fluorescence detection. The sensor we used could contribute to early and rapid detection of potential presence of OTA in wine samples.
2008

abstract No: 

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Full conference title: 

9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
    • ECFG 9th (2008)