Real-time PCR/DNA melting curve-based assay to identify individual strongylid larvae recovered from ovine faecal cultures

A closed-tube real-time PCR method was developed to identify individual nematode larvae recovered from ovine faecal cultures. The method builds on an earlier conventional PCR approach established by our group and similarly targets species-specific sequence motifs in the second internal transcribed spacer region (ITS-2) of ribosomal DNA. However, the new procedure employs real-time PCR together with melting curve analyses to identify species-specific amplicons, thus avoiding the need to undertake gel electrophoresis to differentiate between the species. As with the earlier conventional method, the new assay involves two sets of species-specific reactions. The first is designed to identify Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum venulosum while the second identifies Trichostrongylus axei, Trichostrongylus vitrinis, Cooperia curticei, and Chabertia ovina. With two exceptions, the DNA primers employed in the new assay are identical to those used in the conventional method. The two exceptions are the forward “generic” (conserved) primer, which was re-designed to generate smaller amplicon sizes more suitable for melting curve analysis, and the T. axei-specific primer, which was modified by the addition of a 12 nucleotide GC clamp to the 5' end to achieve a higher melt temperature to enable larvae of this species to be more readily differentiated from those of C. curticei. The melt temperature range for amplicons representing each of the species targeted by the assay was determined using lysates derived from microscopically identified adult male worms (2-12 / species), as well as a total of 30 larvae of each species derived from at least 6 different geographical locations within New Zealand. Results confirmed that there was at least a 0.4oC difference between the top and bottom of the melt temperature range for each of the species. This new assay provides a simpler, higher throughput method to identify individual strongylid nematode L3’s than the earlier conventional PCR assay.