Qualitative, quantitative and molecular detection of aflatoxins from maize grains in north-west India

Thirty three isolates of Aspergillus flavus collected from different maize growing areas from Indian Punjab were screened for aflatoxin production using qualitative and quantitative methods. Qualitatively, bioassay on okra re- vealed 11 isolates to be positive for aflatoxin production with chlorosis index ranging from 0.14 to 0.33. The isolate Af 4 produced highest chlorosis index (1.33) thereby causing maximum reduction in germination of okra seedlings (20%). Concurrently, ammonia vapour test however, revealed 6 isolates positive for aflatoxin production, with one isolate Af 29 being highly toxigenic showing dark red colour development. In total, twelve isolates of A. flavus were observed to be positive using qualitative detection methods. These were further subjected to quantitative (HPLC) and molecular analysis. Out of these 12, eight isolates produced aflatoxin B (AFB ) in the range of 89 - 3269.2 µg kg-1, with highest concentration of AFB detected in Af 5 sample of Ludhiana district. These isolates were confirmed positive using aflatoxin gene specific primers OmtB, Omt1 and Nor1. Sequence analysis of the Nor1 gene amplified from six isolates with different levels of toxin production showed high homology among themselves. The amino acid sequence analysis showed that protein sequences were highly conserved with 99% similarity among them and with other strains in database