Kaplan R M, Dame J B, Reddy G R, Courtney C H
Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32611-0880, USA.
Int J Parasitol. 1995 May;25(5):601-10. doi: 10.1016/0020-7519(94)00159-l.
Epizootiologic studies on F. hepatica frequently use microscopic techniques for the detection of infected snails, however, the poor efficiency, sensitivity, and specificity associated with these techniques limit their usefulness. A DNA-based test for the identification of snails infected with larval stages of F. hepatica would solve these problems and enable a level of detection accuracy previously unavailable. We have cloned and sequenced a 124 bp fragment of repetitive DNA from F. hepatica which hybridizes specifically with DNA of F. hepatica but not with DNA of its snail intermediate hosts Fossaria cubensis and Pseudosuccinea columella, or with DNA of Fascioloides magna and Paramphistomum liorchis, ruminant trematodes which share the same intermediate host and same enzootic range as F. hepatica. Using this 124 bp fragment as a probe, infection in snails was detected immediately following miracidial penetration, thus a sensitivity equivalent to the minimum biologic unit of the parasite was achieved. This 124 bp repeated sequence belongs to a large family of 124 bp repeats that share a high level of sequence identity and constitute approximately 15% of the F. hepatica genome. We also report here the development of a quick and inexpensive DNA extraction protocol for use in field-collected snails. Thus, we have developed both a highly sensitive and specific DNA probe and a means to use the probe in a large epizootiologic study of F. hepatica where thousands of field-collected snails need to be assayed for infection.
肝片吸虫的流行病学研究经常使用显微镜技术来检测受感染的蜗牛，然而，这些技术效率低下、灵敏度和特异性较差，限制了它们的实用性。一种基于DNA的检测方法，用于鉴定感染肝片吸虫幼虫阶段的蜗牛，将解决这些问题，并能实现前所未有的检测准确性。我们已经克隆并测序了一段来自肝片吸虫的124bp重复DNA片段，它能与肝片吸虫的DNA特异性杂交，但不与它的蜗牛中间宿主古巴小土蜗和柱形伪琥珀螺的DNA杂交，也不与巨片形吸虫和梨形双腔吸虫的DNA杂交，这两种反刍动物吸虫与肝片吸虫共享相同的中间宿主和相同的动物疫区范围。使用这个124bp片段作为探针，在毛蚴穿透后立即检测到蜗牛感染，从而达到了与寄生虫最小生物单位相当的灵敏度。这个124bp重复序列属于一个由124bp重复序列组成的大家族，它们具有高度的序列同一性，约占肝片吸虫基因组的15%。我们还在此报告了一种用于野外采集蜗牛的快速且廉价的DNA提取方案的开发。因此，我们开发了一种高度灵敏和特异的DNA探针，以及一种在肝片吸虫的大型流行病学研究中使用该探针的方法，在该研究中需要对数千只野外采集的蜗牛进行感染检测。
