Development of a multiplex real-time PCR to quantify aflatoxin, ochratoxin A and patulin producing molds in foods.

ABSTRACT

A multiplex real-time PCR (qPCR) method to quantify aflatoxin, ochratoxin A (OTA) and patulin producing molds in foods was developed. For this, the primer pairs F/R-omt, F/R-npstr and F/R-idhtrb and the TaqMan probes, OMTprobe, NPSprobe and IDHprobe targeting the omt-1, otanpsPN and idh genes involved in aflatoxin, OTA and patulin biosynthesis, respectively, were used. The functionality of the developed qPCR method was demonstrated by the high linear relationship of the standard curves constructed with the omt-1, otanpsPN and idh gene copies and threshold cycle (Ct) values for the respective producing molds tested to quantify aflatoxin, OTA and patulin producing molds. The ability of the optimized qPCR protocol to quantify producing molds was evaluated in different artificially inoculated foods (fruits, nuts, cereals and dry-ripened meat and cheese products). Efficiency values ranged from 81 to 110% in all inoculated foods. The detection limit was between 3 and 1logcfu/g for aflatoxin, OTA and patulin producing molds. The developed multiplex qPCR was shown be an appropriate tool for sensitive quantification of growth of toxigenic fungi in foods throughout the incubation time. Thus, the multiplex qPCR is a useful, rapid and efficient method to quantify simultaneously aflatoxin, OTA and patulin producing molds in food products.