Multiplex Real-Time PCR Detection of Fumonisin-Producing and Trichothecene-Producing Groups of Fusarium Species

Abstract

Some species of Fusarium can produce mycotoxins during food processing procedures that facilitate fungal growth, such as the malting of barley. The objectives of this study were to develop a 5' fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusarium spp. and to identify Fusarium graminearum and Fusarium verticillioides in field-collected barley and corn samples. Primers and probes were designed from genes involved in mycotoxin biosynthesis (TRI6 and FUM1), and for a genus-specific internal positive control, primers and a probe were designed from Fusarium rDNA sequences. Real-time PCR conditions were optimized for amplification of the three products in a single reaction format. The specificity of the assay was confirmed by testing 9 Fusarium spp. and 33 non-Fusarium fungal species. With serial dilutions of purified genomic DNA from F. verticillioides, F. graminearum, or both as the template, the detection limit of the assay was 5 pg of genomic DNA per reaction. The three products were detectable over four orders of magnitude of template concentration (5 pg to 5 ng of genomic DNA per reaction); at 50 ng template per reaction, only the TRI6 and FUM1 PCR products were detected. Barley and corn samples were evaluated for the presence of Fusarium spp. with traditional microbiological methods and with the real-time PCR assay. The 20 barley samples and 1 corn sample that contained F. graminearum by traditional methods of analysis tested positive for the TRI6 and internal transcribed spacer (ITS) PCR products. The five corn samples that tested positive for F. verticillioides by traditional methods also were positive for the FUMI and ITS PCR products. These results indicate that the described multiplex real-time PCR assay provides sensitive and accurate differential detection of fumonisin- and trichothecene-producing groups of Fusarium spp. in complex matrices.

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

“…PCR assays developed for Fusarium species were reviewed by Nicholson et al (2003). More recently, real-time quantitative PCR assays with fluorogenic sequence-specific probes for fusaria have been developed (Bluhm et al, 2004;Reischer et al, 2004;Waalwijk et al, 2004;Klemsdal et al, 2006;Yli-Mattila et al, 2006). Real-time PCR provides a means for detection and quantification of DNA targets by monitoring PCR product accumulation during the thermal cycling as indicated by increased fluorescence.…”

Genus-Specific Primers for Study of Fusarium Communities in Field Samples

“…A common method for detect-ing and quantifying Fusarium in field samples is real-time PCR using species-specific primers (28). Multiplex real-time PCR has been used for the simultaneous detection of multiple Fusarium species (29,30), but detection is still limited to a few species. Description of Fusarium diversity without targeting specific species is traditionally performed by isolation on selective media, and identification is based on morphological and/or molecular characteristics, but this is a time-consuming procedure (27,31).…”

Impact of aggressiveness of Fusarium graminearum and F. culmorum isolates on yield parameters and mycotoxin production in wheat

“…The primer and probes used to detect the members of the genus Fusarium (ITS assay) as well as the trichothecene producing group of Fusarium (TRI 6 assay) have been reported previously (Bluhm et al 2004). Using the TRI 6 assay, the concentration of DNA encoding trichothecenes (TRI-DNA) was determined and expressed in ng TRI-DNA per g dry matter of wheat heads.…”