Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas (CSIC), Avda. Américo Vespucio s/n, 41092, Seville, Spain.
Parasitol Res. 2011 Jun;108(6):1513-7. doi: 10.1007/s00436-010-2209-z. Epub 2010 Dec 18.
The present study aimed to establish a fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Based on the sequences of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA, we designed a set of genus-specific primers for the amplification of Fasciola ITS-2, with an estimated size of 140 bp. These primers were labelled by fluorescence dyes, and the PCR products were analyzed by capillary electrophoresis under non-denaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments displayed three different peak profiles that allowed the accurate identification of Fasciola species: one single peak specific for either Fasciola hepatica or Fasciola gigantica and a doublet peak corresponding to the "intermediate" Fasciola. Validation of our novel method was performed using Fasciola specimens from different host animals from China, Spain, Nigeria, and Egypt. This F-PCR-SSCP assay provides a rapid, simple, and robust tool for the identification and differentiation between Fasciola spp.
本研究旨在建立一种基于荧光聚合酶链反应-单链构象多态性（F-PCR-SSCP）的方法，用于鉴定片形吸虫属。基于核核糖体 DNA 的第二内部转录间隔区（ITS-2）的序列，我们设计了一组用于扩增 Fasciola ITS-2 的属特异性引物，预计大小为 140bp。这些引物用荧光染料标记，PCR 产物在非变性条件下通过毛细管电泳进行分析（F-PCR-SSCP）。荧光标记 DNA 片段的毛细管电泳分析显示了三种不同的峰型，可准确鉴定片形吸虫属：一种特异性的肝片形吸虫或巨片形吸虫的单峰，以及与“中间”片形吸虫相对应的双峰。使用来自中国、西班牙、尼日利亚和埃及不同宿主动物的片形吸虫标本对我们的新方法进行了验证。这种 F-PCR-SSCP 检测方法为鉴定和区分片形吸虫属提供了一种快速、简单、可靠的工具。
