ABSTRACT
Direct DNA extraction methods, as fi rst performed by Torsvik et al. in 1980, have opened the route to new molecular-based applications including the direct detection of pathogens in biological fl uids or the characterization of the diversity of uncultivable microfl ora. These methods have been combined with signal amplifi cation procedures such as polymerase chain reaction (PCR) to characterize fungi in particular habitats without the need for enrichment or isolation. Being recognized as a rapid, sensitive, and specifi c molecular diagnostic tool, PCR can be extremely effective with pure nucleic acids, with the capability of generating detectable signal from a single copy of target template. However, its sensitivity may be reduced dramatically when applied directly to biological (clinical, environmental, and food) samples. This is due mainly to the fact that many foodstuff, clinical, and environmental samples harbor substances that inhibit or reduce the amplifi cation capacity of PCR. The PCR inhibitors may act on one or more of the following ways by inactivation of the thermostable
DNA polymerase, by degradation or capture of the nucleic acids, or by interfering with the cell lysis step. The optimization of PCR testing conditions has been used to improve the amplifi cation capacity of the DNA polymerase, but in most cases, a sample preparation step is required prior to PCR. Thus, reliable and sensitive detection of the target fungal organisms from the complex samples is dependent on the abililty of sample processing procedures to recover and extract nucleic acids of adequate quantity and quality from these samples. Consequently, much effort is being devoted to the development of sample preparation methodologies that yield PCR-compatible templates from fungal samples; and from the vast number of procedures and articles being published on this topic to date, it appears that the problems associated with molecular detection of fungal pathogens directly from uncultured samples are still far from being solved.
