Black Aspergilli are second most important cause of contamination of poultry feed and their by-products with ochratoxin-A after Aspergillus ochraceous in Pakistan. A total of 23 black Aspergilli were isolated from poultry feeds. Of these black Aspergilli samples, 12 (52.17%) were toxigenic (Ochratoxin A). A PCR based molecular method of cloning and sequencing of ITS (internal transcribed spacer) regions of fungal DNA was developed to identify these fungi.  A universal pair of primers (ITS1 and ITS4) was used and PCR product (600 bp) for Aspergillus niger was amplified. These specific universal primers of ITS region were tested for twelve DNA samples of different isolates of Aspergillus species isolated from poultry feeds. The sequencing of ITS-region provided a powerful and reliable tool for identification of black Aspergilli responsible for the production of ochratoxin-A in poultry feed.

Key words: Aspergillus niger, DNA, ITS region, Ochratoxin A, PCR