Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
Iran J Basic Med Sci. 2013 Dec;16(12):1266-9.
OBJECTIVE(S): Fascioliasis is a zoonotic parasitic disease caused by liver fluke species of Fasciola hepatica and Fasciola gigantica. Differentiation of these two species, based on their morphological characteristics, is difficult. The current study aimed to use PCR-RFLP assay to distinguish between F. hepatica and F. gigantica, based on profiles of RFLP, produced by effect of endonucleases on ITS2 of the ribosomal DNA genes from these two species.
Adult Fasciola spp. were isolated from bile duct of naturally infected animals. The species of Fasciola were confirmed by sequencing the 505 bp region of the ITS2 gene in the isolates. By running the sequences of the samples in NEBcutter, suitable restriction enzymes (MspI and KpnI) were selected. Eight F. gigantica and eighteen F. hepatica samples were evaluated.
While RFLP pattern with MspI produced a profile by which it was difficult to differentiate these two species, KpnI along with MspI, produced a consistent pattern of a 231, 212 and 93 bp fragments in F. hepatica. This pattern was not seen in F. gigantica.
Findings of this study demonstrated that RFLP with KpnI and MspI produce a suitable pattern which simply differentiates F. hepatica from F. gigantica.
片形吸虫病是一种由肝片形吸虫和巨片形吸虫引起的人畜共患寄生虫病。基于形态特征对这两种物种进行区分很困难。本研究旨在使用 PCR-RFLP 分析，根据核糖体 DNA 基因 ITS2 中内切酶对这两种物种产生的 RFLP 图谱，来区分肝片形吸虫和巨片形吸虫。
从自然感染动物的胆管中分离出成虫期片形吸虫。通过对分离株 ITS2 基因的 505 bp 区域进行测序，确认片形吸虫的种类。通过在 NEBcutter 中运行样本序列，选择合适的限制酶（MspI 和 KpnI）。评估了 8 个巨片形吸虫和 18 个肝片形吸虫样本。
虽然 MspI 的 RFLP 图谱难以区分这两种物种，但 KpnI 与 MspI 一起产生了肝片形吸虫中 231、212 和 93 bp 片段的一致图谱。在巨片形吸虫中未观察到这种模式。
本研究结果表明，KpnI 和 MspI 的 RFLP 产生了一种简单区分肝片形吸虫和巨片形吸虫的合适模式。
