SINGLE-STRAND CONFORMATION POLYMORPHISM (SSCP)-BASED MUTATION SCANNING APPROACHES TO FINGERPRINT SEQUENCE VARIATION IN RIBOSOMAL DNA OF ASCARIDOID NEMATODES

In this study, we assessed single-strand conformation polymorphism (SS CP)-based approaches for their capacity to fingerprint sequence variat ion in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/ or human health significance. The second internal transcribed spacer r egion (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual p arasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, To xascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced a llowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for wh ich multiple samples were available, the method allowed the direct dis play of four distinct sequence types of ITS-2 among individual worms o f T. carl. Comparison of SSCP/sequencing with the methods of dideoxy f ingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence typ es, whereas REF displayed three of four. The findings indicate the use fulness of the SSCP-based approaches for the identification of ascarid oid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerp rint microheterogeneity in ITS-2 rDNA using such approaches also has i mplications for studying fundamental aspects relating to mutational ch ange in rDNA.