Polymerase chain reaction-linked single-strand conformation polymorphism of ribosomal DNA to fingerprint parasites.

Abstract

To overcome limitations of the morphological identification of some parasites (and their different developmental stages) to species, we have established a polymerase chain reaction-linked single-strand conformation polymorphism technique (PCR-SSCP) utilizing the second internal transcribed spacer (ITS-2) of ribosomal (r)DNA. This spacer was specifically chosen in the study of strongylid and ascarid nematodes because it is known to provide reliable species markers. The ITS-2 from individual parasites was amplified by PCR, then denatured and subjected to electrophoresis on a mutation detection enhancement (MDE) (nondenaturing) gel matrix. PCR-SSCP analysis showed that the single-strand ITS-2 patterns produced allowed the accurate identification of species. The method also allowed the display of (low-level) variation in patterns within some species between different geographical isolates. These findings demonstrate the usefulness of PCR-SSCP of ITS-2 for the identification of nematode species and indicate its potential for resolving variation in the ITS-2 sequence within species.