Kato Hirotomo, Uezato Hiroshi, Katakura Ken, Calvopiña Manuel, Marco Jorge D, Barroso Paola A, Gomez Eduardo A, Mimori Tatsuyuki, Korenaga Masataka, Iwata Hiroyuki, Nonaka Shigeo, Hashiguchi Yoshihisa
Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Am J Trop Med Hyg. 2005 Jan;72(1):87-93.
The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.
对流行地区利什曼原虫和白蛉种类进行监测对于预测利什曼病的风险和传播范围十分重要。在本研究中，我们开发了一种基于聚合酶链反应（PCR）的方法来检测单个白蛉体内的利什曼原虫微小环DNA。使用该方法，我们在183只白蛉中的6只（3.3%）检测到微小环DNA，而在同一地区通过显微镜检查，143只白蛉中有5只（3.5%）利什曼原虫前鞭毛体呈阳性。通过细胞色素b基因的核苷酸测序，这些物种被鉴定为墨西哥利什曼原虫（Leishmania (Leishmania) mexicana）。此外，通过对PCR扩增的18S核糖体RNA基因片段进行限制性酶切分析，所有利什曼原虫阳性的白蛉均被鉴定为阿亚库乔罗蛉（Lutzomyia ayacuchensis）。由于这种联合方法相对简便且能处理大量样本，它将成为快速鉴定流行地区白蛉和利什曼原虫种类以及监测白蛉群体感染率的有力工具。
