de Morais Rayana Carla Silva, da Costa Oliveira Cintia Nascimento, de Albuquerque Suênia da Cunha Gonçalves, Mendonça Trajano Silva Lays Adrianne, Pessoa-E-Silva Rômulo, Alves da Cruz Heidi Lacerda, de Brito Maria Edileuza Felinto, de Paiva Cavalcanti Milena
Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil.
Federal University of Pernambuco, Cidade Universitária, Av. Moraes Rego s/n, CEP 50670-420, Recife-PE, Brazil.
Exp Parasitol. 2016 Jun;165:43-50. doi: 10.1016/j.exppara.2016.03.005. Epub 2016 Mar 9.
Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques.
qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains.
Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing and qPCR (p = 0.2566). In contrast, a highly significant difference was observed for qPCR and RFLP (p < 0.001).
In this study, we demonstrated the potential use of qPCR as a tool for Leishmania species identification using two Tm ranges.
皮肤利什曼病（CL）是由多种利什曼原虫引起的寄生虫病。多项研究表明，实时定量聚合酶链反应（qPCR）可通过分析熔解温度（Tm）用于利什曼原虫属的鉴定。因此，本研究的目的是评估qPCR区分引起相同临床疾病形式的八种密切相关利什曼原虫的可行性，并将结果与传统技术进行比较。
使用参考菌株进行标准化Tm的qPCR检测。在家畜血液样本进行CL诊断后，通过Tm分析阳性样本，并对qPCR产物进行纯化和测序。还通过Tm分析了10份先前通过多位点酶电泳（MLEE）鉴定的人类样本。还使用参考菌株对限制性片段长度多态性（RFLP）检测（一种参考检测方法）进行了标准化。
通过对利什曼原虫属的Tm进行标准化，创建了两个用于分析的Tm范围：范围1（Tm = 78 - 79.99°C）包括巴西利什曼原虫（Viannia）、巴拿马利什曼原虫（Viannia）、莱氏利什曼原虫（Viannia）、圭亚那利什曼原虫（Viannia）和沙氏利什曼原虫（Viannia）；范围2（Tm = 80 - 82.2°C）包括奈菲利什曼原虫（Viannia）、亚马逊利什曼原虫（Leishmania）和墨西哥利什曼原虫（Leishmania）。共分析了223份阳性血液样本，其中58份在范围1，165份在范围2。通过测序鉴定出巴西利什曼原虫（Viannia）、巴拿马利什曼原虫（Viannia）和圭亚那利什曼原虫（Viannia），通过RFLP分析鉴定出巴西利什曼原虫（Viannia）、墨西哥利什曼原虫（Leishmania）和巴拿马利什曼原虫（Viannia）。还通过qPCR Tm分析了10份先前通过多位点酶电泳（MLEE）鉴定的人类样本；5份分类在范围1，5份在范围2。计算出qPCR与金标准（MLEE）之间的一致性为80%，两种方法之间无显著差异（p = 0.6499）；测序和qPCR也观察到类似结果（p = 0.2566）。相比之下，qPCR与RFLP之间观察到高度显著差异（p < 0.001）。
在本研究中，我们证明了使用两个Tm范围，qPCR作为利什曼原虫物种鉴定工具的潜在用途。
