Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction

Abstract

This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the β-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic T _m-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC®-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.

Keywords:

• Aspergillus carbonarius
• Aspergillus niger aggregate
• Aspergillus tubingensis
• grapes
• mycotoxin
• ochratoxin A
• polyketide synthase
• real-time polymerase chain reaction
• wine

Acknowledgements

The authors are grateful to Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT) Projects (Nos AGL2005-00707 and AGL2004-03060) for financial support. M.V.S. is grateful to the CSIC (Spanish National Research Council) for an I3P Contract (No. I3PDR-7-01). P.V.M.C. is supported by a Ramón y Cajal research contract, co-financed by the Spanish Government and the University of Valencia.