The ribosomal RNA gene cluster occurs in fungi as tandem repeats of a structured unit comprising three ribosomal RNA subunit genes, internal transcribes spacers (ITS) and intergenic spacers (IGS) (Figure 2). The DNA sequences within the subunits in general contain some extremely conserved sequences useful for the development of broad-spectrum primers that allow amplification of fungal sequences from mixed DNA samples. The resulting fragments can be further fractionated to obtain species or genus specific profiles by other techniques, e.g., nested PCR, SSCP, hybridization, ELOSA, RFLP analysis, or sequencing. The more variable spacer regions can be used for genus- or species-specific detection approaches.
There are several reports where 18S rDNA sequences have been used for the detection and identification of fungi. Kappe et al. (1996), Smit et al. (1999), and Borneman and Hartin (2000) developed primer pairs based on this region for the
detection of a wide range of fungi. Mayer (2002) also developed a primer pair and a Taqman probe based on 18 S rDNA sequences, which was used successfully for real-time PCR detection of fungi in black pepper, red pepper, corn, and cereal samples. Makimura et al. (1994) developed a PCR detection system based on 18S rDNA sequences for detection of Aspergillus and Penicillium species. Cappa and Cocconcelli (2001) developed an 18 S rRNA-based assay for the detection of fungi in food samples.
Although the DNA coding for the large (28S) ribosomal rRNA subunit is relatively conserved and is more commonly used for work at higher taxonomic levels, certain portions of it, particularly the eukaryotic D1 and D2 divergent domains near the 5' end are variable enough to detect species-specific differences. The D1-D2 region has extensively been used for phylogenetic studies of aspergilli, penicillia, and yeasts as well as other fungi (Peterson 2000; Rigo et al. 2001). Universal 28S rDNA-based primers were developed by Sandhu et al. (1995). These authors used species-specific probes in post-PCR hybridization reactions to detect the presence of fungi of interest even in situations containing mixed fungal species.
The ITS region has been most frequently used as target for species-specific detection of fungi in foods and feeds. This region was targeted for the detection of spoilage yeasts including Zygosaccharomyces sp. and Torulaspora delbrueckii (Sancho et al. 2000), Saccharomyces cerevisiae (Arlorio et al. 1999), Alternaria sp. (Zur et al. 1999), Penicillia (Pedersen et al. 1997; Boysen et al. 2000), and Fusarium avenaceum (Schilling 1995). Olsson (2000) applied a QC-PCR approach using ITS-based primers for the detection of Penicillium species including Penicillium roquefortii in cereals. Grimm and Geisen (1998) developed ITS-based primer pairs for the detection of fumonisin producing Fusarium species. The sensitivity of the assay was increased using PCR-ELISA. Hendolin et al. (2000) developed a panfungal PCR technique coupled with multiplex liquid hybridization based on ITS specific primers for the detection of a number of fungi in clinical specimens.
Although the ITS region allows for discrimination of closely-related species, it may not be sufficiently variable to distinguish sibling biological species or isolates. The intergenic spacer region of the nuclear rDNA (IGS) however, has been used for species determination when ITS regions lacked sufficient variation (Spreadbury et al. 1993). Ribosomal RNAs are also encoded in the mitochondria and have been used for molecular identification (Wakefield et al. 1990). Fulton and Brown (1997) developed a primer pair based on sequences of an intron located within the 18 S rRNA gene for the detection of Monilinia fructicola. Montone and Litzky (1995) targeted the 5 S rRNA gene for the detection of different Aspergillus species.
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