Molecular identification and differentiation of fasciola isolates using PCR-RFLP method based on internal transcribed spacer [ITS1, 5.8S rDNA, ITS2]

ABSTRACT

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species. The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety tematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp409I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates. The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates