Quantitative analysis of ITS2 sequences in trichostrongyle parasites

Real-time PCR assays were developed to identify and quantify common gastrointestinal nematodes of ruminants. The assays were based on genus-specific primer and probe combinations derived from the second internal transcribed spacer (ITS2) ribosomal DNA transcription unit of Haemonchus contortus, Ostertagia leptospicularis, Trichostrongylus colubriformis and Cooperia curticei. The TaqMan probes for the first two species were labelled with 6-carboxyfluorescein and those for the latter two with VIC and all were synthesised as dihydrocyclopyrroloindole minor groove binder conjugates. Cloned ITS2 DNA or genomic DNA isolated from first stage larvae, derived from overnight cultures, were used as template. The real-time PCRs reproducibly allowed the identification of at least 100 copies of cloned ITS2 DNA or one hundredth part of a single larval genomic DNA equivalent. The assays proved to be genus-specific since the addition of DNA from heterologous trichostrongyle genera did not change the cycle threshold values obtained with only homologous DNA. Furthermore, the use of genomic DNA of several other ruminant nematode parasite genera gave negative results. In duplex experiments, 6-carboxyfluorescein-labelled H. contortus or O. leptospicularis probes were used together with the VIC-labelled T. colubriformis and C. curticei probes, respectively, confirming the specificity and sensitivity of the probes found in the simplex experiments. The primer and probe combinations gave comparable results when applied with core reagents from different suppliers and with both the M x 4000 and the ABI 7700 instruments. This technique provides means for a rapid, reliable and quantitative detection and differentiation of the most important parasitic nematodes of sheep and cattle.