Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids
Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids
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DOI：
10.1016/j.parint.2010.11.005
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发表时间：
2011-01-01
影响因子：
1.9
通讯作者：
Oku, Yuzaburo
中科院分区：
文献类型：
--
作者：
Armua-Fernandez, Maria Teresa;Nonaka, Nariaki;Oku, Yuzaburo
我们报告了一种针对线粒体基因NADH脱氢酶亚基1（nad 1）的比色PCR/斑点印迹法用于带绦虫卵的鉴别诊断。对绦虫nad 1基因的部分序列进行了比对，并根据保守区设计了新的引物。然后根据保守引物之间的可变区，人工设计犬带绦虫种特异性寡核苷酸探针（S-SONP）。具体而言，S-SONP是针对粗头绦虫、T. Hydatigena、T. multiceps，T. ovis，T.带形棘球绦虫、细粒棘球绦虫（基因型1）、E. multilocularis和E.沃杰利。每个探针显示出高特异性，因为没有观察到与任何扩增的nad 1片段的交叉杂交。我们使用49个绦虫卵阳性样品收集的狗在赞比亚进行了评估。每个样品中提取5 - 10个卵的DNA。应用PCR/斑点杂交技术，成功地检测到了T. 42份样品中有棘球绦虫T.在3个样品中为多头菌，在其余4个样品中为两种菌（混合感染）。结果表明，PCR/斑点杂交试验是一个可靠的替代鉴别诊断带虫卵粪便样品。(C)2010爱思唯尔爱尔兰有限公司版权所有。
We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples. (C) 2010 Elsevier Ireland Ltd. All rights reserved.