Detection of Aspergillus flavus in maize kernels by conventional and real-time PCR assays

Aflatoxins are carcinogenic secondary metabolites mainly produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops, such as maize, groundnut, cotton and chillies. Aflatoxin contamination is considered one of the most serious food safety issues worldwide. To secure the safety of food, regular monitoring of the levels of aflatoxins in foods and feeds is necessary. An alternative could be the detection of the aflatoxin-producing fungi in foods and feeds. In this study a polymerase chain reaction (PCR) method was developed for rapid, specific and sensitive detection of A. flavus in maize kernels. A. flavus-specific primers based on the O-methyltransferase gene (omt-A) that is involved in the aflatoxin B1 biosynthesis, were designed and used to detect the fungus by PCR. The designed primers were highly specific to A. flavus. The molecular detection sensitivity of A. flavus was 1 ng of purified fungal DNA template in conventional PCR. A real-time PCR assay was standardized for rapid, specific and sensitive detection of A. flavus in corn kernels by using these primers. The standard curve obtained showed a linear correlation between copy number of the cloned target DNA sequence of A. flavus and Cycle threshold (Ct) values, with R² of 0.98. These PCR-based assays may be highly useful in food and feed industries and quarantine laboratories for detection of this fungus.