Hybridization Based Methods

In its basic application, DNA or RNA is fixed to a solid phase and a labeled probe is added and allowed to react with its complementary sequence (Southern 1975). The direct hybridization techniques used for the detection of fungi include in situ hybridization and colony or dot blot hybridization methods (Geisen 1998; Sterflinger et al. 1998). Besides DNA probes, peptide nucleic acid (PNA) probes have also been developed. Peptide nucleic acids are pseudopeptides in which the sugar phosphate backbone of DNA is replaced by a polyamide backbone. Due to the uncharged backbone, PNA probes have unique properties relative to oligonucleotides. These characteristics include faster hybridization kinetics, and the ability to form a stable PNA/NA hybrid even at low ion concentrations, necessary to disrupt the secondary structure of nucleic acids such as rRNA (Perry-O'Keefe et al. 2001a,b; Stender et al. 2001).

A recent development of hybridization-based techniques is the microarray technology. DNA microarrays are glass slides containing an ordered mosaic of the entire genome as a collection of either oligonucleotides (oligonucleotide micro-arrays) or PCR products representing individual genes (cDNA microarrays). The development of microarrays has been fuelled by the application of robotic technology to routine molecular biology. This technique allows a single hybridization to be performed against multiple replicates of a single microbial genome, or against copies of several unrelated genomes on a single glass slide. Microarrays could be used to identify virulence genes as PCR products in food-borne bacteria (Chizhikov et al. 2001). Similarly, they could also be used for the direct detection of a variety of fungi contaminating foods and feeds.

Hybridization techniques have only rarely been used for direct detection of fungi in food matrices due to the lack of demanded sensitivity (Scheu et al. 1998). Kosse et al. (1997) developed 18 S rDNA-based probes for the detection of yogurt spoilage yeasts, while Geisen (1998) developed suitable probes for the detection of fumonisin producing Fusarium species based on randomly selected sequences. Chemiluminescent DNA probe kits are available for the detection of some medically important fungi including Blastomyces dermatitidis, Histoplasma capsulatum, Cocci-dioides immitis, and Cryptococcus neoformans (GenProbe, San Diego, USA). However, to our knowledge, such kits have not yet been developed for food-borne fungi.

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