Leishmania protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several Leishmania species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that L. (L.) infantum and L. (L.) amazonensis can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in L. (L.) infantum and L. (L.) amazonensis, respectively. This paper describes the application of this approach to L. (L.) mexicana and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from L. (L.) amazonensis. We show that L. (L.) mexicana can be distinguished from L. (L.) infantum using the same approach we had previously validated for L. (L.) amazonensis. Moreover, it was also possible to reliably discriminate L. (L.) mexicana from L. (L.) amazonensis by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate L. (L.) infantum, L. (L.) amazonensis, L. (L.) mexicana and Viannia subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.

中文翻译：

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利什曼原虫（L.）婴儿，利什曼原虫（L.）amazonensis和墨西哥利什曼原虫（L.）mexicana的区分使用顺序qPCR分析和高分辨率熔解分析。

利什曼原虫是内脏，皮肤和粘膜皮肤利什曼病的病原体。在特定的地理区域，例如拉丁美洲，几种利什曼原虫是地方性的并且同时存在。因此，需要一种用于物种歧视的诊断方法。在这种尝试中，已经开发了许多基于qPCR的测定法。最近，我们已经表明，L.（L.）婴儿和L.（L.）亚马孙可以通过CQ值从两个qPCR测定（定量PCR-ML和qPCR-AMA），设计成比较区分AMPLIFY kDNA小环亚类在婴儿L.（L.）和亚马逊L.（L.）中更具代表性， 分别。本文介绍了该方法在墨西哥乳杆菌中的应用，并介绍了一种新的qPCR-ITS1检测方法，然后进行高分辨率熔体（HRM）分析，以区分该物种与亚马逊乳杆菌。我们显示，可以使用我们之前为亚马逊L.（L.）验证的相同方法，将墨西哥L.（x。）区别于婴儿L.（L.）。此外，也有人能够可靠地判别L.（L.）墨西哥从L.（L。）亚马孙通过使用qPCR-ITS1，然后进行HRM分析。因此，建立了基于加上HRM分析顺序qPCR测定的诊断算法以识别/分化L.（L.）婴儿，L.（L.）亚马孙，L.（L.）墨西哥和Viannia亚属。这些发现更新并扩展了我们研究小组发布的先前数据，从而为具有共存物种的流行地区提供了额外的诊断工具。