ORIGINAL ARTICLE Detection of aflatoxigenicity of Aspergillus flavus, based on potential gene marker, from food and feed samples Saima Khalid | Noshal Hussain | Muhammad Imran Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan Correspondence Dr. Muhammad Imran, Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islama- bad 45320, Pakistan. Email: m-imran766@hotmail.com or mmimran@qau.edu.pk Abstract In this study, the incidence of aflatoxigenic strains of Aspergillus flavus was evaluated in cattle feed, water, and milk from three districts of Northern Punjab region of Pakistan. In total, 39 of the hun- dred samples collected were found contaminated by A. flavus. Specifically, the incidence of A. flavus was 54.28 %, 53.3 %, and 11.42 % in feed (n 5 35), water (n 5 30), and milk (n 5 35) samples, respectively. The UV method indicated aflatoxigenic potential in 62 % of strains, and the ammonia vapor test showed 54 % of samples to be positive for aflatoxin production. Polymerase chain reac- tion detection of aflatoxin producing cluster of A. flavus was done by identifying four structural genes, that is, nor-1, ver-1, omt-A, afl-R. It is inferred that omt-A and afl-R genes were regarded as potential markers for aflatoxins production. Therefore, this study proved that omt-A could be used for rapid detection of potential aflatoxigenic strains in feed, water and food products. Practical applications In this study, the isolates of Aspergillus flavus were investigated for their aflatoxigenic nature using cultural (fluorescence under UV-light and NH 4 OH vapor induced color change test) and molecular (PCR) methods. The UV test is more sensitive than the ammonia test; however, comparable results from both methods strengthened our confidence in the findings. PCR results indicated the omt-A and afl-R genes as potential markers for aflatoxins biosynthesis because these genes were ampli- fied in all those strains that were regarded as aflatoxigenic based on cultural methods for aflatoxin detection. Thus, the combined use of cultural and molecular methods used in this study can pro- vide a cheaper and faster way to detect aflatoxigenic A. flavus in food and feed samples in developing countries. 1 | INTRODUCTION The study of Turkey-X disease led toward aflatoxin discovery in 1960 (Blount, 1961). Aflatoxins are prominent toxins of food supply. Afla- toxin contamination in food and feed is caused by invasion of Aspergil- lus flavus and Aspergillus parasiticus that are chief species involved in aflatoxin production. The Aspergillus family is not fastidious and can grow on rotten leaves in soil. This can cause an Aspergillus invasion dur- ing harvest, and contamination during processing or storage of food and feed. Animal feed directly affects the food chain; therefore, feed should be free of nutritional contaminants, particularly mycotoxins, to secure animal and ultimately consumer’s health (Arroyo-Manzanares, Huertas-Perez, Gamiz-Gracia, & Garcia-Campana, 2015). Conventional targets for Aspergillus contamination are staple food and feed items. Millions of losses in the poultry and dairy industry are caused due to contamination by this fungus. Because it can grow at environmental temperature on plant debris and organic waste materials, the fungus can adapt to different ecologies and contaminate food commodities, making aflatoxins a ubiquitous threat for the food chain. Ingestion of aflatoxin contaminated food can cause many acute and chronic disorders. Recurrent observations of aflatoxin exposure are immunosuppression, hepatic carcinoma, liver cirrhosis, edema, and hemorrhage (Uppala, 2011). Aflatoxin exposure during pregnancy can cause faltering growth in infants, and aflatoxins ingestion can cause growth retardation in young mammals (Turner, Subrahmanyam, & Pilet- sky, 2009). However, adult mammals usually show higher tolerance lev- els. High health hazards posed by aflatoxin for human health made it a suitable agent for biological warfare. External parameters such as tem- perature, humidity, and intergranular gas composition directly affects aflatoxin biosynthesis (Abdel-Hadi, Carter, & Magan, 2011) and they J Food Saf. 2018;e12448. https://doi.org/10.1111/jfs.12448 wileyonlinelibrary.com/journal/jfs V C 2018 Wiley Periodicals, Inc. | 1 of 9 Received: 31 August 2017 | Revised: 3 December 2017 | Accepted: 1 January 2018 DOI: 10.1111/jfs.12448