Other Sequences As Targets

One approach used for developing suitable species- or strain-specific probes for the detection of fungi is based on the random amplified polymorphic DNA technique (RAPD) (Williams et al. 1990). The RAPD is a variation of conventional PCR where one primer of arbitrary sequence is used, and the annealing temperature is low (usually 35°C). Species- or strain-specific RAPD fragments are selected, sequenced, and suitable primers are devised to amplify the specific fragment in conventional PCR reactions. Such SCAR markers have been successfully used to develop species-specific probes for a number of Fusarium species (Chelkowski et al. 1999; Nicholson et al. 1996; Nicholson et al. 1998; Schilling et al. 1996; Young et al. 2001), particularly fumonisin-producing fusaria (Geisen 1998), and for Aspergillus fumigatus (Brandt et al. 1998; Varga et al. 2002, unpublished results). Murillo et al. (1998) developed a primer pair based on the sequence of a random genomic clone for the detection of Fusarium moniliforme.

For the detection of Fusarium graminearum, another target, the galactose oxidase (gaoA) gene has been used (Niessen and Vogel 1997). This enzyme is produced by only a limited number of fungi including F. graminearum, Gibberella fujikuroi, and Beltraniella portoricensis. Niessen et al. (1998) also developed a solid phase PCR with detection of immobilized amplified product in a one-phase system (DIAPOPS) to specifically amplify and quantify a DNA fragment of the gaoA gene from F. graminearum.

Microsatellite-based probes were developed for the detection of Epichloe species in different grasses (Groppe and Boller 1997). Mayer (2002) developed a Penicillium nordicum specific primer pair and a Taqman probe based on a polyketide synthase gene sequence for the detection of this species in cereal samples. Pearson and McKee (1992) developed a multiplex PCR method based on plasmid sequences for the detection and discrimination of S. cerevisiae, Zygosaccharomyces bailii, and Z. rouxii in foods. Pearson et al. (1995) designed a PCR-based assay to detect retrotransposon long terminal repeat elements for the detection of Pichia membranefaciens.

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