Abstract

Aspergillus flavus is the causal agent of corns and peanuts mold known to produce aflatoxin. A quick and reliable PCR-based diagnostic assay has been developed to detect A. flavus using a fungus-specific marker derived from genomic DNA. An amplified RAPD product of 600 bp obtained in A. flavus isolates using a random primer OPB-11 was cloned in pGEMT easy vector and sequenced. Based on sequences, six primers were designed, out of which a primer pair Asp f1 (CCCGTGAAGTTGCCCAGGT) and Asp r2 (GTCGTTTGGTGAGTGGGAA) amplified a sequence of 490 bp which was specific toA. flavus. The specificity of the marker when tested against 31 isolates of Aspergillus flavus, 7 isolates of Aspergillus clavatus, 4 each isolates of Aspergillus terreus, Aspergillus oryaze, Aspergillus tamari, 1 each isolate of A. parasiticus and A. kambarensis and 4 isolates of 2 different Cheatomium species, 5 isolates of 3 differentTrichoderma species and 5 isolates of 5 different Fusarium species showed a specific band of 600 bp only in A. flavus. With the optimized PCR parameters, this sequence characterized amplified region (SCAR) marker was sensitive and could detect small quantities of A. flavus DNA as low as 10 to 25 ng with high efficiency. This marker could also clearly distinguish A. flavus from other fungal plant pathogens, including differentAspergillus spp. The utilization of this diagnostic PCR assay in analysis of post harvest samples will be a strong step towards aflatoxin detection in animal feed and export commodity.

Key words: Aspergillus flavus, OPB11, random amplified polymorphic DNA (RAPD), sequence characterized amplified region (SCAR) marker