Amplification of the second internal transcribed spacer ribosomal DNA of individual trichostrongylid nematode larvae by nested polymerase chain reaction

Abstract

The second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) is used as a genetic marker to identify trichostrongylid nematodes. However, it is often difficult to amplify by polymerase chain reaction (PCR) the ITS2 rDNA of a single trichostrongylid nematode larva or egg. A nested PCR (nPCR) assay was, therefore, developed to amplify the ITS2 from individual trichostrongylid nematode larvae. The results show that the ITS2 rDNA of a significantly greater proportion of individual larvae was amplified using nPCR compared with a standard PCR. There was also no need to column-purify the genomic DNA before nPCR, which is more time and cost effective for studies involving large sample sizes. The amplicons produced from the secondary phase of the nPCR were subjected to single-strand conformation polymorphism analyses and DNA sequencing to confirm the species identity of the larvae used in the current study as Ostertagia gruehneri. The nPCR assay was also used to amplify the ITS2 from individual trichostrongylid eggs. The ability to amplify the ITS2 rDNA from large numbers of individual nematode eggs and larvae has important implications for diagnostic testing and for conducting epidemiological studies on these parasites of veterinary importance.