Aspergillus species, particularly these belonging to section Nigri, are responsible for ochratoxin A (OTA) contamination of table and wine producing grapes worldwide. Aspergillus carbonarius is considered the most potent OTA producer among the other Aspergilli. In order to monitor contamination and prevent OTAentering the food chain it is crucial to have a rapid and specific method that would detect and quantifypotential OTA-producing species at the early stages of infection. Detection of these fungi in grapes is particularly critical around harvest time, when the risk of contamination and OTA production is considered elevated.
Conventional culturing techniques have been used for identification and quantification of these fungi since many years. However, these methods are time-consuming, require multiple steps and often are not able to provide accurate results for fungal identification. Characterization of black Aspergillus species based on morphological keys usually requires taxonomic expertise and quantification may be biased due to growth rate differences between species on grapes.
The development and evolution of molecular biology techniques such as the Polymerase Chain Reaction (PCR) and real-time PCR have provided new approaches for fungal detection and quantification on foodstuffs through detection and quantification of their DNA. PCR-based methods have provided alternatives to conventional techniques in order to detect potentially toxigenic molds. Both techniques are based on the selective amplification of DNA sequences that can serve as specific molecular markers for a species.
Conventional PCR (end point PCR) is suitable for detection of the species under question in a food sample or a complex matrix, and can be also used in combination with other techniques such as Restriction Fragment Length Polymorphism (RFLP) or Multilocus Sequencing for characterization of a fungal species. Real-time PCR (or quantitative PCR) allows detection and quantification of the target species in a food sample, and monitoring of the results can be done while the reaction proceeds (in real time). The working principle and chemistries used in real-time PCR confer greater specificity and sensitivity to the technique with respect to conventional PCR. Furthermore quantitative PCR can be used for studying the expression of selected gene targets. Both techniques offer the possibility to work in a multiplex mode, allowing the simultaneous amplification of more than one target sequence in a single reaction.
Under the scope of monitoring and predicting grape contamination by OTA producing fungi, a considerable number of end-point and real-time PCR assays targeting various genes or genomic regions from different Aspergillus and Penicillium species have been developed. A. carbonarius specific methods developed so far target either the ITS region, calmodulin or a pks gene whose expression was reported to be related to OTA production. The same genomic regions were used as targets for the development of A. carbonarius specific SYBR Green and/or TaqMan probe real-time PCR assays as well.
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