Multiplex Real-Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera
Article first published online: 20 DEC 2012
© 2012 Institute of Food Technologists®
Journal of Food Science
Volume 78, Issue 1, pages M70–M76, January 2013
How to Cite
Vegi, A. and Wolf-Hall, C. E. (2013), Multiplex Real-Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera. Journal of Food Science, 78: M70–M76. doi: 10.1111/j.1750-3841.2012.03008.x
- Issue published online: 9 JAN 2013
- Article first published online: 20 DEC 2012
- MS 20120577 Submitted 4/23/2012, Accepted 10/23/2012.
- real-time PCR
Abstract: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real-time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene-producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON- and OTA-producing fungi can be detected and quantified in a single reaction tube using this multiplex real-time PCR method.
Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal-based foods.