Ghatee Ma, Sharifi I, Mirhendi H, Kanannejad Z, Hatam G
Dept. of Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran ; Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
Iran J Parasitol. 2013 Apr;8(2):264-72.
Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs.
Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products.
Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis.
Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.
热带利什曼原虫是一个基因分化的物种。对从伊朗巴姆地区（伊朗人兽共患皮肤利什曼病（ACL）的著名疫源地之一）获得的热带利什曼原虫分离株的整个内部转录间隔区（ITS）进行扩增，在琼脂糖凝胶电泳中显示出双带模式。本研究解释了这种模式是如何产生的。
从伊朗东南部的巴姆地区收集了27份热带利什曼原虫涂片标本，从伊朗西南部的设拉子收集了8份硕大利什曼原虫和1份婴儿利什曼原虫涂片标本。此外，使用整个ITS-PCR对1份硕大利什曼原虫和1份婴儿利什曼原虫培养标准菌株进行检测，以观察其电泳模式。对已存入GenBank的热带利什曼原虫、硕大利什曼原虫和婴儿利什曼原虫的ITS序列进行了分析。对热带利什曼原虫的GenBank序列分析显示，根据长度大小可分为两组序列，一组有100 bp的缺口。因此，设计了一种新的反向引物LITS-MG，以排除PCR产物中的这一缺口。
在所有热带利什曼原虫病例中，整个ITS片段扩增均产生双带模式，而婴儿利什曼原虫和硕大利什曼原虫分离株则观察到清晰的单带。这一结果与从GenBank序列的电子分析中获得的结果一致。使用LITS-MG引物预期会为热带利什曼原虫产生一条包含ITS1、5.8s和部分ITS2产物的单带，这适用于后续的分子研究，如测序或限制性分析。
对GenBank热带利什曼原虫序列的分析以及随后的实际实验室检测显示，热带利什曼原虫中至少有两个等位基因，这在巴姆分离株中得到了证实。这种特殊的双带模式是由于ITS-rDNA等位基因内存在100 bp的片段差异。
