Development of Two Quantitative Real-Time PCR Methods Based on SYBR Green and TaqMan to Quantify Sterigmatocystin-Producing Molds in Foods

摘要

Sterigmatocystin (ST) is a mycotoxin produced by different species of molds that can grow and contaminate some foods such as dry-ripened foods, cereals, and spices. To improve food safety, the presence of ST-producing molds in foods should be quantified. In the present work, two real-time quantitative PCR (qPCR) protocols based on SYBR Green and TaqMan were developed. Twenty-eight ST-producing and non-producing strains belonging to different species usually reported in food products were used. The STproduction of the reference strains was checked by micellar electrokinetic capillary electrophoresis and high-performance liquid chromatography–mass spectrometry. For development of the qPCR methods, a primer pair (FluGF1/FluGR1) and a TaqMan probe (FluGp) were designed on the basis of fluG gene, involved in the ST biosynthesis. The presence of fungal DNA was tested by using a qPCR method based on the universal fungal β-tubulin gene. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the fluG gene copies number and Ct values for the different ST producers tested. The ability to quantify ST producers of the developed SYBR Green and TaqMan assays in different artificiallyinoculated food samples and naturally infected samples was successful, with a minimum detection limit of 1 log cfu/g. In conclusion, both qPCR methods developed allow quantifying ST-producing molds, in raw materials/ingredients and pre-processed foods, which they would be very useful for monitoring these toxigenic molds in HACCP programs, to prevent ST accumulation in processed foods.