Ceccarelli Marcello, Galluzzi Luca, Diotallevi Aurora, Andreoni Francesca, Fowler Hailie, Petersen Christine, Vitale Fabrizio, Magnani Mauro
Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino, PU, Italy.
Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, IA, USA.
Parasit Vectors. 2017 May 16;10(1):239. doi: 10.1186/s13071-017-2181-x.
Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area.
DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed.
The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the C values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of C values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples.
A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.
利什曼病是一种由多种利什曼原虫引起的被忽视的疾病，这些利什曼原虫属于利什曼原虫亚属（利什曼原虫）和利什曼原虫亚属（维氏亚属）。已经报道了几种基于qPCR的分子诊断方法用于利什曼原虫种类的检测和定量。这些方法中的许多都使用动基体DNA（kDNA）小环作为靶序列。由于利什曼原虫种类之间的序列相似性，这些检测存在潜在的跨物种扩增。先前的研究通过基于kDNA设计的SYBR绿基qPCR检测，随后进行熔解或高分辨率熔解（HRM）分析，证明了利什曼原虫亚属（利什曼原虫）和利什曼原虫亚属（维氏亚属）之间的区分。重要的是，这些方法不能完全区分婴儿利什曼原虫和亚马逊利什曼原虫，它们可以在同一地理区域共存。
来自18株婴儿利什曼原虫、亚马逊利什曼原虫、巴西利什曼原虫、巴拿马利什曼原虫、圭亚那利什曼原虫的菌株/分离物的DNA以及来自感染婴儿利什曼原虫的犬的62份临床样本通过先前开发的qPCR（qPCR-ML）进行扩增，并进行HRM分析；使用ABI PRISM 310遗传分析仪对选定的PCR产物进行测序。基于获得的序列，设计了一种新的SYBR绿基qPCR检测（qPCR-ama），旨在扩增在亚马逊利什曼原虫中更丰富的小环亚类。
qPCR-ML随后进行HRM分析在53.4%的病例中无法区分亚马逊利什曼原虫和婴儿利什曼原虫。因此，对新的基于SYBR绿的qPCR（qPCR-ama）进行了测试。该检测在掺入宿主DNA的样本中实现了0.1 pg寄生虫DNA的检测限，并且未显示与克氏锥虫或宿主DNA的交叉扩增。尽管qPCR-ama也扩增了婴儿利什曼原虫菌株，但与qPCR-ML相比，C值显著增加。因此，对qPCR-ML和qPCR-ama的C值进行联合分析可以在100%的测试样本中区分婴儿利什曼原虫和亚马逊利什曼原虫。
开发了一种新的、经济实惠的基于SYBR绿qPCR的方法来区分婴儿利什曼原虫和亚马逊利什曼原虫，该方法利用了小环序列的主要丰度，而不是针对假设的物种特异性序列。这些物种之间快速准确的区分对于提供适当的预后和治疗可能是有用的。
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