Department of Clinical Science, Faculty of Medicine, University of Bergen, Postboks 7804, 5020, Bergen, Norway.
Department of Physiology, Institute of Biosciences, University of São Paulo, Rua do Matão Travessa 14 no. 101, São Paulo, SP, 05508900, Brazil.
Parasit Vectors. 2018 Jul 16;11(1):421. doi: 10.1186/s13071-018-2989-z.
The leishmaniases comprise a spectrum of clinical manifestations caused by different species of Leishmania. Identification of species is important for diagnosis, treatment and follow-up management. However, there is no gold standard for species identification. High resolution melting analysis (HRM) offers a possibility to differentiate Leishmania species without the need for processing of the PCR-product. The amino acid permease 3 (aap3) gene is an exclusive target for trypanosomatids and is conserved among Leishmania spp., thus it can be a valuable target for an HRM assay for diagnosis of the leishmaniases.
The HRM dissociation profiles of three amplicons targeting the aap3-coding region allowed the discrimination of L. (Leishmania) donovani, L. (L.) infantum, L. (L.) major, L. (L.) tropica, L. (L.) mexicana, L. (L.) amazonensis, L. (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) naiffi and L. (V.) shawi using DNA from promastigote cultures. The protocol was validated with DNA samples from clinical infection in humans and a cat, naturally infected sand flies, and experimentally infected mice.
HRM analysis using the aap3 coding sequence as target is a relatively cheap, fast and robust strategy to detect and discriminate Leishmania species from all the endemic regions worldwide. The target and method proved to be useful in clinical, field and experimental samples, thus it could be used as a tool in diagnosis as well as ecological and epidemiological studies.
利什曼病是由不同种利什曼原虫引起的一系列临床表现。种属鉴定对诊断、治疗和随访管理都非常重要。然而，目前还没有物种鉴定的金标准。高分辨率熔解分析（HRM）提供了一种无需对 PCR 产物进行处理即可区分利什曼原虫种属的可能性。氨基酸通透酶 3（aap3）基因是原生动物的特有靶标，并且在利什曼原虫属中是保守的，因此它可以作为 HRM 检测用于诊断利什曼病的一个有价值的靶标。
针对 aap3 编码区的三个扩增子的 HRM 解离曲线允许区分利什曼原虫（L.）donovani、L.（L.）infantum、L.（L.）major、L.（L.）tropica、L.（L.）mexicana、L.（L.）amazonensis、L.（Viannia）braziliensis、L.（V.）guyanensis、L.（V.）lainsoni、L.（V.）naiffi 和 L.（V.）shawii，所用 DNA 来自前鞭毛体培养物。该方案用来自人类和猫的临床感染、自然感染的白蛉以及实验感染的小鼠的 DNA 样本进行了验证。
使用 aap3 编码序列作为靶标的 HRM 分析是一种相对廉价、快速且稳健的策略，可用于检测和区分来自全球所有流行地区的利什曼原虫种属。该靶标和方法在临床、现场和实验样本中都很有用，因此可用于诊断以及生态和流行病学研究。
