title: Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal.
abstract: The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6 gene involved in trichothecene biosynthesis and the FUM5 gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearum and at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearum and its differentiation from F. verticillioides were accomplished prior to visible fungal growth at <10(5) CFU/g of cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industry's effort to monitor quality and safety.
authors.affiliation: Department of Botany and Plant Pathology, Purdue University, 1155 Lilly Hall, West Lafayette, Indiana 47907, USA.
authors.forename: B H
authors.initials: BH
authors.lastname: Bluhm
authors.forename: J E
authors.initials: JE
authors.lastname: Flaherty
authors.forename: M A
authors.initials: MA
authors.lastname: Cousin
authors.forename: C P
authors.initials: CP
authors.lastname: Woloshuk
mesh_terms: D003257:Consumer Product Safety; D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics; D021901:DNA, Intergenic; D005516:Food Microbiology; D037341:Fumonisins; D005670:Fusarium / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D009183:Mycotoxins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014255:Trichothecenes / Q000096:biosynthesis / Q000737:chemistry; D003313:Zea mays / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021901:DNA, Intergenic; D037341:Fumonisins; D009183:Mycotoxins; D014255:Trichothecenes