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

使用非放射性 DNA 探针鉴定南美利什曼原虫寄生虫种类

• 批准号: 03670204
• 负责人: ESHITA Yuki
• 金额: $ 1.28万
• 依托单位: Kurume University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670204/
• 关键词: Leishmania parasite; Sandfly; Southern blot hybridization; Oligonucleotide primer; Leishmania mexicana; Leishmania braziliensis; Non-radioactive probe; Polymerase chain reaction (PCR); polymerase chain reaction(PCR); リーシュマニア症; polymerase chain r

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.

对三组合成的寡核苷酸引物进行了聚合酶链反应（PCR）试验。第1组引物只扩增巴西疟原虫复合体（L.braziliensis、L.panamensis和L.guyanensis）的DNA，第2组引物除扩增墨西哥疟原虫复合体（L.amazonensis、L.pifanoi、L.garnhami）外，只扩增墨西哥疟原虫复合体的DNA，第3组引物扩增墨西哥疟原虫连同WHO参考菌株的所有其他列希曼原虫DNA。其中3组应用于已鉴定的厄瓜多尔利什曼原虫酶解分析。59株未鉴定的厄瓜多尔分离物中，50株经第1组鉴定为巴西锥虫复合寄生虫，2株经第2、3组鉴定为墨西哥锥虫，5株经第1、2、3组引物及形态特征鉴定为锥虫。不到10个寄生虫的数量足以用PCR扩增特定的DNA产物。非放射性地高辛- dna探针的Southern blot杂交技术支持利什曼原虫在分子水平上的鉴定。研制了蛇痘卢佐菌特异性DNA探针，引物的构建正在进行中。上述结果表明，利用这3组引物进行PCR可将巴西利什曼复合体、墨西哥利什曼复合体和墨西哥利什曼复合体与其他利什曼复合体，尤其是锥虫进行区分，可用于南美利什曼病的流行病学调查。

项目成果

Eshita, Y: "Detection of Leishmania parasites by DNA amplification using polymerase chain reaction (PCR)" Studies on New World leishmaniasis and its transmission, with particular reference to Ecuador, Research Report Series No.3 (Y.Hashiguchi, ed), Kyowa

Eshita, Y：“使用聚合酶链式反应 (PCR) 通过 DNA 扩增检测利什曼原虫寄生虫”关于新世界利什曼病及其传播的研究，特别是厄瓜多尔，研究报告系列第 3 号（Y.Hashiguchi，编辑），Kyowa

Alexander,J.B.: "New records of phlebotomine sand flies(Diptera:Psychodidae)from Ecuador" Mem.Inst.Oswardo Cruz,Rio de Janeiro. 87. 123-130 (1992)

Alexander, J.B.：“来自厄瓜多尔的白蛉（双翅目：Psychodidae）的新记录”Mem.Inst.Oswardo Cruz，里约热内卢。

橋口,義久: "エクアドル高地のアンデス型リーシュマニア症(Andean Ieishmaniasis)" 日熱医会誌. 21. 41 (1993)

Hashiguchi, Yoshihisa：“厄瓜多尔高地的安第斯伊什曼病”日本发热医学会杂志 21. 41 (1993)。

Eshita, Y: "Diagnosis of trypanosomiasis with the parasite specific DNA probes - Non-radioactive probes and PCR methods -" Nippon rinsho. 50 (Spetial edition). 480-485 (1992)

Eshita, Y：“用寄生虫特异性 DNA 探针诊断锥虫病 - 非放射性探针和 PCR 方法 -”Nippon rinsho。

16-21 (1992)

16-21 (1992)