Advances in Food Mycology

80 Ai Lin Beh et al.
DNA in PCR amplicons (Kiesling et al., 2002) (Table 3). Speciesspecific
primers are used in PCR assays to generate amplicons.
Production of the amplicon means that the particular target species
is present in the sample. Qualitative detection of the target amplicon
is done by its visualisation in gel electrophoresis. Real time PCR systems
are now being applied to yeasts, and allow the simultaneous
detection and quantification of the target species, omitting the electrophoresis
step (Table 3).
Nucleic acid probes and specific primers have gained widespread
use in the detection of bacterial species. Their application to the detection
of yeast species has not been that extensive and further development
is needed. Most probes reported to date have been developed
around specific sequences in ribosomal DNA, and it would be worthwhile
to identify other species-specific genes that could be targeted for
probe development.
2.4. Differentiation of Strains Within a Species
The distinctive character and appeal of many foods and beverages
(eg. bread, beer, wine) produced by fermentation with yeasts are frequently
attributable to the contribution and properties of particular
strains. Strain typing is also useful to trace the source of yeast contamination
in outbreaks of food spoilage. The ability to differentiate
strains within a species is, therefore, an important requirement in quality
assurance programs. Over the past 20 years, a diversity of molecular
methods has been developed and applied to the differentiation of
yeast strains, and some of these are sufficiently robust and convenient
for routine use (Table 4).
Electrophoretic karyotyping of genomic DNA using pulse field
gel electrophoresis (PFGE) and RFLP analysis of genomic DNA
have been widely applied to “fingerprint” yeast strains with very
good success and confidence, but they require significant attention
to DNA preparation and extraction, as well as to subsequent electrophoretic
analyses (Cardinali and Martini, 1994; Deak, 1995; van
der Aa Kühle et al., 2001). Analysis of mitochondrial DNA by
RFLP produces fragment profiles that give excellent strain discrimination.
Simplified methods for extraction and processing of the
mitochondrial DNA have greatly improved the convenience and
reliability of this assay, and consequently it has found significant
application to the analysis of food and beverage yeasts (Querol
et al., 1992; López et al., 2001; see review of Loureiro and Malfieto-
Ferreira, 2003).