Sequence analysis of the β-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples
Sequence analysis of the β-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples
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DOI：
10.1016/s0020-7519(02)00068-1
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发表时间：
2002-07-01
影响因子：
4
通讯作者：
Pozio, E
中科院分区：
文献类型：
--
作者：
Cacciò, SA;De Giacomo, M;Pozio, E
鞭毛虫寄生虫贾第鞭毛虫是全世界人类和动物腹泻的主要原因。分子技术对于研究分类学、种群结构、动物分离株的人畜共患病潜力以及寄生虫的遗传变异性与在人类中观察到的临床症状范围之间的相关性特别有用。在这项工作中，一种新的PCR检测，靶向β-贾第虫基因进行了测试，21贾第虫贾第虫参考菌株代表汇编A，B和E，这是与人类和其他哺乳动物的感染。然后将该测定应用于从意大利人收集的30个粪便样品。31个PCR产物的序列分析表明，每个组合是明显不同的基础上，特定的取代;序列的多样性约为5%，和所有的取代发生在第三个密码子的位置的基因。组合内变异性的分析允许在组合A内鉴定三种基因型，在组合B内鉴定四种基因型。有趣的是，两种基因型仅在临床样品中鉴定，而在参考菌株中未鉴定。最后，一个简单的PCR-限制性片段长度多态性方法的开发，用于快速判别的组合和应用于直接遗传分析的囊肿存在于人类粪便样品。(C)2002年澳大利亚寄生虫学学会有限公司由爱思唯尔科技有限公司出版。保留所有权利。
The flagellate parasite Giardia duodenalis is a major cause of diarrhoea in humans and in animals worldwide. Molecular techniques are particularly useful for studying the taxonomy, the population structure, the zoonotic potential of animal isolates, and the correlation between the genetic variability of the parasite and the range of clinical symptoms observed in humans. In this work, a new PCR assay that targets the beta-giardin gene was tested on 21 Giardia duodenalis reference strains representing Assemblages A, B and E, which are associated with infections of humans and other mammals. The assay was then applied to 30 faecal samples collected from Italian persons. The sequence analysis of 31 PCR products from both reference strains and clinical samples showed that each Assemblage is clearly distinct from the others on the basis of specific substitutions; the sequence diversity was approximately 5%, and all substitutions occurred at the third codon positions of the gene. The analysis of the intra-Assemblage variability allowed for the identification of three genotypes within Assemblage A, and of four genotypes within Assemblage B. Interestingly, two genotypes were identified only in the clinical samples and not in reference strains. Finally, a simple PCR-restriction fragment length polymorphism method was developed for the rapid discrimination of Assemblages and applied for the direct genetic analysis of cysts present in human faecal samples. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.