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首页> 外文期刊>Journal of Clinical Microbiology >Single-Step Multiplex PCR Assay for Characterization of New World Leishmania Complexes
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Single-Step Multiplex PCR Assay for Characterization of New World Leishmania Complexes

机译：单步多重PCR分析法表征新世界利什曼原虫复合物

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We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, andLeishmania 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 theLeishmania 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 Leishmaniaparasites, particularly where different Leishmaniacomplexes are found in the same geographical area.
机译：我们已经开发出一种PCR方法，可用于一步法区分新世界（em> Leishmania （ Leishmania braziliensis ， Leishmania mexicana 和 >利什曼原虫donovani ）。该多重分析法针对这些生物的剪接前导RNA（微型外显子）基因重复序列，可以同时检测所有三种复合物，从而为每种复合物生成不同大小的产物。该测定法特定于 Leishmania 属，不能识别相关的动素体原生动物，例如 Trypanosoma cruzi ， Trypanosoma brucei 和 Crithidia fasciculata 。它正确地识别了中美洲和南美洲地理分布广泛的利什曼原虫物种。 PCR扩增的灵敏度范围为1 fg至10 pg DNA（0.01至100寄生虫），具体取决于检测到的复合物。简单地通过煮沸稀释的培养物即可制备出的寄生虫粗提物，是扩增的极佳模板。还测试了临床材料的粗制制剂。该测定法检测到了 L。皮肤利什曼病病变的皮肤刮中的巴西巴西人，非典型皮肤利什曼病的皮肤刮ing中的＜Leishmania chagasi 和 L。病灶中的mexicana 从受感染的仓鼠中吸出。我们最小化了材料要求，并最大程度地提高了该测定法的简便性，快速性和信息含量，使其适合在利什曼病流行的国家的实验室中使用。该测定法对于快速鉴定Leishmania 寄生虫，特别是在同一地理区域发现不同的 Leishmania 复合物特别有用。

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