استفاده از روش Fast PCR جهت تشخیص گونه های فاسیولا

Background and purpose: Fasciola hepatica and Fasciola gigantica are common liver flukes that are the etiological agents of fasciolosis, which affects both domestic livestock and humans worldwide. In the present study we established a rapid, easy and also accurate tool, for differentiation between F. hepatica and F. gigantica using Fast PCR. Material and methods: Thirty adults of Fasciola species were isolated from sheep and cattle liver form abattoirs in Mazandaran province. ITS1 rDNA region were amplified by Sapphire Amp® Fast PCR and compared with AccuPower® Taq PCR PreMix from Bioneer. In addition, PCR-RFLP assay using Tsp509I was performed for identification of F. hepatica and F. gigantica. Results: A fragment of approximately 463bp was amplified in all of the Fasciola samples using Sapphire Amp® Fast PCR premix in just 34 minutes, while nucleic acid amplification was completed in about 1 hour and 46 minutes by Bioneer PCR master mix. All PCR products were digested with restriction enzyme TasІ (Tsp509I). After digestion, F. hepatica revealed two fragments of 151 and 312 bp while F. gigantica produced three fragments of 93, 151, and 219 bp. Conclusion: Fast PCR reaction using Sapphire Amp® Fast PCR premix was completed three times faster than the time of conventional premix. The new Fast PCR assay using Sapphire Amp® Fast PCR premix provides a simple, rapid and accurate technique for identification and differentiation of Fasciola species in epidemiological researches on human and domestic animals in endemic regions of fasciolosis.