Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

ABSTRACT Background: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. Methods: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. Results: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. Conclusions: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite’s DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

摘要
背景：皮肤利什曼病（Cutaneous Leishmaniasis）作为一种感染性被忽视疾病，其发病率正逐年攀升。目前针对该病已开发出多种传统诊断方法及常规聚合酶链式反应（PCR）技术，通过靶向不同基因实现利什曼原虫的虫种鉴定。
方法：本研究采集利什曼原虫样本，提取基因组DNA并设计新型特异性高灵敏度引物；采用靶向ITS-rDNA与Cyt b基因的实时定量PCR（qPCR）结合高分辨率熔解（High-Resolution Melting）技术鉴定利什曼原虫，绘制并比对标准曲线，通过高分辨率熔解曲线分析完成虫种鉴定。
结果：在对硕大利什曼原虫（Leishmania major）与热带利什曼原虫（Leishmania tropica）进行分析评价时，ITS-rDNA的熔解温度与阈值循环数均高于Cyt b基因，但Cyt b的灵敏度更优。通过熔解曲线比对、荧光曲线归一化及熔解曲线差异作图，可轻松区分各利什曼原虫虫种：硕大利什曼原虫与热带利什曼原虫的区分温度分别为83.6℃与84.7℃，二者温差小于0.9℃。基于EvaGreen的实时PCR经灵敏度与特异性优化后，可检测至低于1 pmol的DNA浓度。
结论：精准鉴定利什曼原虫对疾病防控策略至关重要。采用EvaGreen的实时PCR可快速、高灵敏且特异地检测寄生虫DNA；经改良的高分辨率熔解技术可生成特征性熔解曲线，并可根据利什曼原虫核苷酸组成的微小差异检测单核苷酸多态性。