A novel snapback primer probe assay for the detection and discrimination of sympatric Haemonchus species using DNA melting analysis

Different sympatric species of Haemonchus parasites infecting ruminants and camels can be distinguished morphologically, but involves tedious microscopic examinations, measurements and several other limitations. Information on internal transcribed spacer-2 (ITS-2) sequence provides confirmatory differentiation of sympatric Haemonchus species. The present study introduces a novel, snapback primer probe based, real time PCR assay for the differentiation of three sympatric Haemonchus species, H. contortus (Hco), H. placei (Hpl) and H. longistipes (Hlo). The assay was designed to amplify a region of 130bp within the ITS-2 gene that included three diagnostic mutational sites capable of discriminating Hco, Hpl and Hlo. Following melt curve analysis, species-specific diagnostic melt peaks were obtained for Hco, Hpl and Hlo with a mean melting temperature of 56.6±0.3°C, 64.4±0.1°C and 54.4±0.1°C respectively. The test for analytical sensitivity revealed the ability of the assay to detect up to 5 copies per reaction. To evaluate the discriminating power of the assay, 174 samples from adult worms and 3rd stage larvae belonging to different Haemonchus species and various other nematode species including Cooperia curticei, Trichostrongylus axei, Trichostrongylus colubriformis, and Teladorsagia circumcincta were tested. Additionally, DNA extracted from 25 fecal egg samples was also tested and the specificity of the assay was verified by sequencing the ITS-2 gene of all the Haemonchus positive and non-Haemonchus samples. The assay worked accurately with 100% specificity in at least three real time PCR platforms. The assay is an effective alternative to the sequencing approach and is expected to be helpful for the screening of individual adult and larval Haemonchus parasites. However, caution needs to be applied while interpreting the results from fecal egg samples due to varying levels of sympatric co-infections from different Haemonchus species. The present study is the first report on the application of snapback primer probe methodology for the differentiation of nematode parasites.