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 trichothecene producing Fusarium species and ochratoxin A producing Penicillium and Aspergillus species in cereal grains.
Primers and probes were designed targeting the Tri5 gene in trichothecene producing Fusarium, rRNA gene in Penicillium verrucosum and polyketide synthase gene in Aspergillus ochraceus. The method was highly specific to the species containing these genes and sensitive, detecting 3 pg of genomic DNA. These products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). Thirty barley samples were evaluated for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi using the above method. Among these samples, 9 tested 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.
Primers and probes were designed targeting the Tri5 gene in trichothecene producing Fusarium, rRNA gene in Penicillium verrucosum and polyketide synthase gene in Aspergillus ochraceus. The method was highly specific to the species containing these genes and sensitive, detecting 3 pg of genomic DNA. These products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). Thirty barley samples were evaluated for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi using the above method. Among these samples, 9 tested 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.
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