Harris E, Kropp G, Belli A, Rodriguez B, Agabian N
Program in Molecular Pathogenesis, University of California, San Francisco, 94143-0422, USA.
J Clin Microbiol. 1998 Jul;36(7):1989-95. doi: 10.1128/JCM.36.7.1989-1995.1998.
We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmania parasites, particularly where different Leishmania complexes are found in the same geographical area.
我们开发了一种聚合酶链反应（PCR）检测方法，用于一步鉴别新大陆利什曼原虫的三种复合体（巴西利什曼原虫、墨西哥利什曼原虫和杜氏利什曼原虫）。这种多重检测方法针对这些生物体的剪接前导RNA（小外显子）基因重复序列，能够同时检测所有三种复合体，为每种复合体生成大小不同的产物。该检测方法对利什曼原虫属具有特异性，不识别相关的动基体原生动物，如克氏锥虫、布氏锥虫和fasiculata隐鞭虫。它正确地鉴定了在中美洲和南美洲广泛地理分布的利什曼原虫物种。PCR扩增的灵敏度范围为1 fg至10 pg DNA（0.01至100个寄生虫），具体取决于检测到的复合体。仅通过煮沸稀释培养物制备的培养寄生虫粗提物，是扩增的优良模板。还对临床材料的粗制品进行了检测。该检测方法在皮肤利什曼病病变的皮肤刮片中检测到巴西利什曼原虫，在非典型皮肤利什曼病的皮肤刮片中检测到恰加斯利什曼原虫，在感染仓鼠的病变吸出物中检测到墨西哥利什曼原虫。我们已将该检测方法的材料需求降至最低，并最大限度地提高了其简易性、快速性和信息量，使其适用于利什曼病流行国家的实验室。该检测方法对于在国内快速鉴定利什曼原虫寄生虫应该是有用的，特别是在同一地理区域发现不同利什曼原虫复合体的地方。
