Laboratory of Medical Entomology, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz/FIOCRUZ, 30190-002 Belo Horizonte, MG, Brazil.
Diagn Microbiol Infect Dis. 2010 Dec;68(4):401-9. doi: 10.1016/j.diagmicrobio.2010.08.007.
Leishmaniasis is a disease caused by the protozoan Leishmania resulting in a variety of clinical manifestations, from self-healing skin lesions to fatal visceral disease. The development of polymerase chain reaction (PCR)-based techniques has made species identification easier, faster, and less labor intensive. The main targets for PCR amplification include kinetoplastid DNA (kDNA), miniexon, and conserved regions such as the internal transcribed spacer. The objective of this work was to evaluate 4 different PCR techniques designed to type Leishmania using laboratory strains. Parasites were subjected to 4 PCR procedures using specific Leishmania primers for miniexon (designated A1 and A2) and kDNA (designated B1 and B2, C1 and C2, and D1, D2 and D3). Discrimination between some species and the 2 main subgenera Leishmania and Viannia was achieved. Unweighted pair group method analysis resulted in the expected clustering of the 2 species from the subgenus Leishmania. However, some species in the subgenus Viannia could not be distinguished, representing a continued challenge for PCR-based protocols. Results are discussed in terms of advantages, limitations, and reproducibility of these 4 PCR-based techniques in the taxonomy of Leishmania.
利什曼病是一种由原生动物利什曼原虫引起的疾病，导致各种临床表现，从自我愈合的皮肤损伤到致命的内脏疾病。聚合酶链反应（PCR）技术的发展使得物种鉴定更容易、更快且劳动强度更低。PCR 扩增的主要目标包括动基体 DNA（kDNA）、小外显子和内转录间隔区等保守区域。本工作的目的是评估 4 种不同的 PCR 技术，用于使用实验室菌株对利什曼原虫进行分型。寄生虫通过 4 种使用针对小外显子（命名为 A1 和 A2）和 kDNA（命名为 B1 和 B2、C1 和 C2 以及 D1、D2 和 D3）的利什曼原虫引物的 PCR 程序进行处理。实现了一些物种和 2 个主要亚属利什曼和 Viannia 之间的区分。非加权对组方法分析导致来自利什曼亚属的 2 个物种按照预期聚类。然而，Viannia 亚属中的一些物种无法区分，这仍然是基于 PCR 的方案的一个持续挑战。根据这些基于 4 种 PCR 技术在利什曼原虫分类学中的优势、局限性和可重复性，对结果进行了讨论。
