In silico and in vitro evaluation of primers for molecular differentiation of Leishmania species

Abstract Leishmaniasis is a zoonotic disease caused by over 20 species of protozoan parasites of the genus Leishmania. Infection is commonly spread by sandflies and produces a wide spectrum of clinical signs and symptoms. Therefore, from an epidemiological and therapeutic standpoint, it is important to detect and differentiate Leishmania spp. The objective of this study was to combinate in silico and in vitro strategies to evaluate the analytical specificity of primers previously described in the literature. According to electronic PCR (e-PCR) analysis, 23 out of 141 pairs of primers selected through literature search matched their previously reported analytical specificity. In vitro evaluation of nine of these primer pairs by quantitative PCR (qPCR) confirmed the analytical specificity of five of them at the level of Leishmania spp., L. mexicana complex or Leishmania and Viannia subgenera. Based on these findings, the combination of e-PCR and qPCR is suggested to be a valuable approach to maximize the specificity of new primer pairs for the laboratory diagnosis of infections with Leishmania spp.

摘要：利什曼病（Leishmaniasis）是由利什曼原虫属（Leishmania）的20余种原生动物寄生虫引发的人畜共患病。该感染通常经白蛉传播，可引发多样化的临床体征与症状。因此，从流行病学与治疗学角度而言，检测并鉴别利什曼原虫属物种（Leishmania spp.）具有重要意义。本研究旨在结合计算机模拟（in silico）与体外（in vitro）策略，评估此前文献中报道的引物的分析特异性。通过电子PCR（e-PCR）分析，在文献检索得到的141对引物中，有23对符合其此前报道的分析特异性。对其中9对引物开展定量PCR（qPCR）体外验证，证实其中5对在利什曼原虫属、墨西哥利什曼原虫复合群（L. mexicana complex）或利什曼亚属与维亚尼亚亚属（Viannia subgenera）层面具备分析特异性。基于上述研究结果，本研究建议将e-PCR与qPCR联用作为一种可靠方法，以最大化用于利什曼原虫属感染实验室诊断的新型引物对的特异性。