Detection of trichothecene producing Fusarium spp. by PCR: adaptation, validation and application to fast food

Abstract

Background. Food contamination by trichothecene mycotoxins is considered to be an emerging public
health problem. The aim of this study was to validate a rapid sonification protocol, previously set up for
cereal Fusarium DNA extraction from fast food samples, produced by a centre for research and development
in the food industry in Catania, Sicily, and to validate it for a diagnostic PCR assay targeted at tri5, the key
gene of trichothecene biosynthesis.
Methods. DNA from reference Fusarium spp. strains and from fast food samples was prepared, setting up an
extraction protocol adapted using some modifications based on a method recently described. Validation
experiments were performed: serial dilution of DNA extracted from fungal cultures were added to food
samples and then DNA was extracted. Specific primer pairs were used to detect F. graminearum and F.
culmorum DNA in species-specific assays as well as trichothecene-producing Fusarium spp. in a group-
specific system.
Results. All genomic DNA extracted from trichothecene-producing Fusarium spp. as well as from DNA-spiked
fast food samples and from food still in it’s original condition resulted in the correct amplification. The
detection limit was 1 x 10-4
μg of DNA. All fungal and food samples tested gave highly consistent results in
repeatability assays, thus demonstrating the within-lab and within/between-day precision of the method.
Conclusions. Information on the epidemiology of trichothecene producing Fusarium through the food chain
and the identification of the most frequently contaminated components of fast food are essential in order to
develop effective public health strategies for minimising consumer exposure to trichothecenes.