Developing a PCR assay to detect Aspergillus parasiticus

Abstract

Aspergillus parasiticus is one of the producers of aflatoxins in food. They may produce aflatoxin B and G which can lead to serious diseases in human. Aflatoxin is known as a type of carcinogens. People ingesting infected food even in a very low level for a long time can get cancer because of aflatoxin accumulation. Detection of aflatoxin or aflatoxin producers in foods is necessary to improve the life quality by decreasing the risk of getting diseases. Recently, the contemporary method to detect A. parasiticus is a morphological method but it still retains many limitations. In this study, a PCR based method was developed to provide a basic for develop a new method to detect A. parasiticus in food that overcame the disadvantages of the conventional morphological method. A specific set of primers was designed based on norB-cypA genes and successfully optimized for best amplification results at 62 ˚C. The sensitivity of the test was identified to be 0.005 ng/µL of target fungi DNA. A DNA isolation protocol was also optimized to ensure the success of the PCR assay, using SDS lysis, sand and thermal shock. The protocol from DNA isolation to PCR was successfully developed and provided a useful tool to improve the diagnosis of aflatoxins at an early stage and control all stages of food production. The success of this study is designing a pair of primers and a PCR assay which is specific for detection of A. parasiticus among other aspergilus species. This PCR assay can be used in the future for further development a PCR method for detection of A. parasiticus in food.