Research Unit in Parasitology and Parasitic Diseases, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium.
Vet Parasitol. 2011 May 31;178(1-2):93-9. doi: 10.1016/j.vetpar.2010.12.020. Epub 2010 Dec 23.
This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex(®) DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react.
本研究旨在开发和验证一种原始的 PCR 方案，以评估在西欧其主要中间宿主圆扁沼螺中存在肝片吸虫的情况。在本研究中，比较了两种 DNA 提取技术，并描述了一种新的多重 PCR 方法。Chelex(®) DNA 提取技术比经典的基于酚/氯仿/蛋白酶 K 的方法更合适，因为它不含有毒有机溶剂，耗时更短、成本更低，并且在 DNA 浓度和波长相位方面具有更高的重现性。多重 PCR 的设置是为了扩增扁卷螺内转录间隔区 2 序列（500-600bp），该序列作为内对照，以及 Fasciola sp. 序列，该序列在整个吸虫基因组中重复超过 300,000 次。96 只蜗牛被混合在一起，其中 6 只蜗牛（6.25%）被检测出 Fasciola sp. 阳性。检测限低于最小生物学感染单位（一个尾蚴）。来自片形吸虫、双腔吸虫和大片吸虫的 DNA 提取物没有交叉反应。
