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Development of a multiplex real-time PCR to quantify aflatoxin, ochratoxin A and patulin producing molds in foods
Alicia Rodríguez1, Mar Rodríguez, María J Andrade
1Food Hygiene and Safety, Faculty of Veterinary Science, University of Extremadura, Avda. de la Universidad s/n., Cáceres, Spain
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.
Insights
A new multiplex real-time PCR method accurately quantifies molds producing aflatoxin, ochratoxin A (OTA), and patulin in foods. This rapid technique offers sensitive detection for improved food safety.
Area of Science:
- Food Microbiology
- Molecular Biology
- Analytical Chemistry
Background:
- Aflatoxins, Ochratoxin A (OTA), and patulin are mycotoxins posing significant risks to food safety.
- Accurate quantification of toxigenic mold populations in diverse food matrices is crucial for risk assessment.
Purpose of the Study:
- To develop and validate a multiplex real-time PCR (qPCR) assay for simultaneous quantification of aflatoxin, OTA, and patulin-producing molds.
- To assess the assay's performance in various food products.
Main Methods:
- Development of specific primer pairs (F/R-omt, F/R-npstr, F/R-idhtrb) and TaqMan probes (OMTprobe, NPSprobe, IDHprobe).
- Targeting genes involved in mycotoxin biosynthesis: omt-1, otanpsPN, and idh.
- Validation using standard curves, efficiency testing (81-110%), and determination of detection limits (1-3 log cfu/g) in inoculated foods.
Main Results:
- High linearity was observed between gene copies and threshold cycle (Ct) values, confirming assay functionality.
- The multiplex qPCR demonstrated high efficiency across various food types (fruits, nuts, cereals, meat, cheese).
- Sensitive detection limits were achieved for all target mold groups.
Conclusions:
- The developed multiplex qPCR method is a sensitive, rapid, and efficient tool for simultaneously quantifying aflatoxin, OTA, and patulin-producing molds in food.
- This assay is suitable for monitoring fungal growth and mycotoxin risk throughout food production and storage.
