Molecular diagnosis of ochratoxinogenic Aspergillus species

Toxigenic and non-toxigenic black aspergilli belonging to the Aspergillus niger aggregate and to A. carbonarius were compared to each other and to strains of other species by DNA fingerprinting. AFLPs showed a clear separation of A. niger and A. carbonarius. However, no clear correlation between the genetic similarity of the strains and the ability to produce ochratoxin A (OTA) was detected. Based on AFLP, marker sequences were chosen for the construction of SCAR-PCR primers for the detection of A. carbonarius. A similar approach was used for A. ochraceus, another fungus of concern regarding ochratoxin A contamination of coffee. Cluster analysis of A. ochraceus isolates mainly from Brazilian coffee showed a very close genetic similarity. Three species specific primer pairs were developed and one of these was used for the PCR and realtime PCR (RT-PCR) based detection of the mould in green coffee. A. ochraceus was specifically and rapidly detected and quantified in green coffee. A positive correlation between the amount of DNA and OTA content was established.