Real-time PCR assay for detection and quantification of Aspergillus carbonarius in grapes: comparison between SYBR®Green and TaqMan®

Amaia González, Belén Patiño, Jéssica Gil-Serna, Covadonga Vázquez, Marí­a Teresa González-Jaén

Author address: 

University Complutense of Madrid, Madrid, Spain

Abstract: 

Aspergillus carbonarius is the main species responsible for the production of Ochratoxin A (OTA) in grapes and wine. This mycotoxin is one of the most common naturally occurring contamination in agroproducts and is toxic to both humans and animals. The purpose of this work was to develop a specific qPCR assay for identifying and quantifying A. carbonarius genomic DNA occurring on contaminated grapes in order to predict the potential OTA risk. Nucleotide sequences obtained in our lab and from the GeneBank were aligned to design a specific primer pair QCARBOF/QCARBOR from the Internal Transcribed Region ITS. A FAM labelled TaqMan® probe was also designed for the TaqMan® assays. The specificity and sensibility of the primer/probe combinations were tested on a number of A. carbonarius strains and on mixes in different proportions of genomic DNA from A. carbonarius and other Aspergillus species commonly found on grapes, Fusarium sp., Penicillium sp. and Alternaria sp. None of the other species alone gave a positive result with this PCR primer set either using Sybr® Green or TaqMan® . No inhibition was even observed in the mixes of different genomic DNAs. To test the ability of the designed primers to detect A. carbonarius in grapes, the QPCR assays were coupled with a fungal enrichment and a DNA extraction method for grapes. Different lots of commercially grapes were contaminated with a spore suspension (102 and 106 spores/ml) and DNA was extracted after 0, 8, 16 and 24 hours of incubation. The results indicated that the critical qPCR amplification product was clearly observed for grapes contaminated by 106 spores without incubation using either SYBR® Green or TaqMan® . These quantitative analyses demonstrated that both reporters SYBR® Green or TaqMan® are equally valid, but we recommend SYBR® Green as is less expensive, easy to use and gave more sensibility than TaqMan® .The qPCR assays presented in this work are based on ITS sequence and therefore are more sensitive than primers based on single copy sequences. Supported by AGL2004-07549-C05-05, AGL2007-66416-C05-02 and UCM-CM-961014.
2008

abstract No: 

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Full conference title: 

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