热休克蛋白 70 PCR-RFLP：新世界和旧世界利什曼原虫种属鉴别通用简单工具。
Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World.
机构信息
Instituto de Medicina Tropical Pedro Kourí, Departamento de Parasitología, La Habana, Cuba.
出版信息
Parasitology. 2010 Jul;137(8):1159-68. doi: 10.1017/S0031182010000089. Epub 2010 May 5.
INTRODUCTION
Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species.
METHODS
Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability.
RESULTS
Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns.
CONCLUSION
Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.
简介
利什曼病的物种分型在诊断、流行病学和临床研究中变得越来越重要。一种基于聚合酶链反应（PCR）扩增子的部分热休克蛋白 70 基因（hsp70）的限制性片段长度多态性（RFLP）分析方法已被用于新大陆，可以鉴定一些物种。
方法
基于我们最近在一项系统发育研究框架内确定的 51 个 hsp70 序列的初步计算机分析，确定了物种特异性的限制性位点。通过对来自 14 个物种的 139 株菌株进行 PCR-RFLP 测试，验证了种内和种间的变异性。
结果
我们的检测方法可以通过进行两次后续消化来鉴定出利什曼原虫、L. donovani、L. tropica、L. aethiopica、L. major、L. lainsoni、L. naiffi、L. braziliensis、L. peruviana、L. guyanensis 和 L. panamensis。L. mexicana、L. amazonensis 和 L. garnhami 没有产生物种特异性的限制性片段模式。
结论
目前没有用于全球利什曼物种鉴别检测的方法。我们提出了一种通用的 PCR-RFLP 方法，可以基于单个 PCR 鉴定出大多数与医学相关的旧大陆和新大陆利什曼原虫物种，避免了进行单独 PCR 的需要。该技术操作简单，可在所有可进行 PCR 的环境中实施。
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