Identification of South American Leishmania parasite-species using non-radioisoopic DNA probes

三套合成的寡核苷酸引物进行了测试的聚合酶链反应（PCR）。第一对引物仅扩增巴西利血吸虫复合群（巴西利血吸虫、巴拿马利血吸虫和圭亚那利血吸虫）的DNA，第二对引物仅扩增墨西哥利血吸虫复合群（亚马逊利血吸虫、皮凡诺利血吸虫和加哈米利血吸虫）的DNA，而不扩增墨西哥利血吸虫;第三对引物扩增墨西哥利血吸虫以及所有其他世界卫生组织参考株的莱希曼原虫DNA。其中三套适用于已经确定的厄瓜多尔利什曼原虫zymode分析。在59个厄瓜多尔分离物中，50个经第一套引物鉴定为巴西锥虫复合寄生虫，2个经第二、第三套引物鉴定为墨西哥锥虫，5个经第一、第二、第三套引物鉴定为其他寄生虫锥虫。用非放射性地高辛标记的DNA探针进行Southern印迹杂交，可在分子水平上对利什曼原虫进行鉴定。本研究开发了阿亚库蚊特异性DNA探针，引物的构建正在进行中。结果表明，利用这3对引物进行的PCR扩增，可将巴西利什曼原虫复合群、墨西哥利什曼原虫复合群和墨西哥利什曼原虫与其它寄生虫，特别是锥虫区分开来，适用于南美洲利什曼病的流行病学调查。

Three sets synthesized-oligonucleotide primers have been tested for the polymerase chain reaction (PCR). The first set of the primers amplified only the DNA of L.braziliensis complex (L.braziliensis, L.panamensis and L.guyanensis), and the second one amplified only L.mexicana complex (L.amazonensis, L.pifanoi, L.garnhami) except L.mexicana, and the third one amplified L.mexicana as well as all other Leshimania parasite DNA of WHO reference strains. Three sets of them were applied for already identified-Ecuadorian Leishmania parasites by zymodeme analysis. Of fifty-nine of unidentified-Ecuadorian isolates, fifty were identified as L.braziliensis complex parasites by the first set, two isolates were identified as L.mexicana by the second and third ones, and the other five seemed to be other parasites, Trypanosoma sp. by the first, second and third ones of the primers as well as morphological features. Less than 10 parasites were enough numbers to amplify the specific DNA products by PCR.Southern blot hybridization using non-radioactive digoxigenin-DNA probes supported the identification of Leishmania parasites in molecular level. Lutzomyia ayacuhensis specific DNA probe was developed and constructionof the primers is in progress. These results indicate that the PCR using the three sets of the primers may differentiate L.braziliensis complex and L.mexicana complex and L.mexicana from others, especially Trypanosoma parasites, and apply for epidemiological survey of leishmaniasis in South America.