Mkada-Driss Imen, Lahmadi Ramzi, Chakroun Ahmed S, Talbi Chiraz, Guerbouj Souheila, Driss Mehdi, Elamine Elwaleed M, Cupolillo Elisa, Mukhtar Moawia M, Guizani Ikram
Laboratoire d'Epidémiologie Moléculaire et de Pathologie Expérimentale (LR11IPT04)/Laboratoire d'Epidémiologie et d'Ecologie Parasitaire (LR00SP04) -Institut Pasteur de Tunis, Université Tunis el Manar, Tunis, Tunisia.
Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan.
PLoS One. 2014 Oct 14;9(10):e109773. doi: 10.1371/journal.pone.0109773. eCollection 2014.
Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5' end transversions, and presence of inter- and intra- taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.
内脏利什曼病（VL）主要由杜氏利什曼原虫复合体引起。VL在包括东非和地中海地区在内的世界许多国家流行，这些地区的流行病学情况复杂。这些病原体的分类存在争议，但它们的遗传多样性与地理起源之间存在关联。随着基因组知识的不断增加，随机扩增多态性DNA（RAPD）仍然是鉴定和表征新型DNA标记的有用方法。我们的目的是使用RAPD鉴定和表征不同地理区域的VL利什曼原虫寄生虫中的多态性DNA标记，以便构建一组有可能区分VL寄生虫的PCR靶标。筛选了100个具有任意DNA序列的不同寡核苷酸十聚体以进行可重复扩增，并选择了28个用于扩增来自12个具有不同起源的杜氏利什曼原虫、阿奇博尔德利什曼原虫和婴儿利什曼原虫菌株的DNA。总共扩增出155条带，其中60.65%表现为多态性。28条引物中有7条提供单态模式。聚类分析允许根据寄生虫的地理起源对其进行聚类。选择差异扩增的条带，其中22个RAPD产物成功克隆并测序。生物信息学分析允许对标记、序列和引物位点进行定位分析。本研究通过Southern杂交进行补充，以确认标记与动基体DNA（kDNA）的归属。生物信息学分析鉴定出16个核标记和3个微小环标记。对这些标记的分析突出了RAPD引物位点的多态性，主要是5'端的颠换，以及分类群间和分类群内复杂序列和微卫星变异的存在；观察到不同序列之间转换与颠换以及插入缺失的偏差，然而在婴儿利什曼原虫和杜氏利什曼原虫之间这种偏差不太明显。该研究提供了一组有充分记录的多态性DNA标记，以开发分子诊断检测方法来表征和区分引起VL的病原体。
