Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran.
Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, Tehran, Iran.
Front Cell Infect Microbiol. 2021 Oct 12;11:731595. doi: 10.3389/fcimb.2021.731595. eCollection 2021.
Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by or ) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig Advanced Standard Kit, a serial dilution of purified plasmid (2-2×10 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for and . The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected and definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions and .
许多实验室方法被用于诊断利什曼病，因为它的症状多种多样，由不同的物种引起。本研究开发并修改了一种基于 TaqMan 探针的定量实时 PCR 方法，用于准确鉴定来自伊朗流行地区的人类皮肤利什曼病（由 或 引起）。考虑了氨基酸渗透酶 3（AAP3）和细胞色素氧化酶 II（COII）的两个基因区域。设计了六组新的种特异性引物和探针。共检查了 123 个样本，并用于评估和验证实时 PCR。根据基因 sig 高级标准试剂盒的寄生虫负荷，在相同条件下，为两个基因制备了纯化质粒（2-2×10 个拷贝/反应）的系列稀释液。特异性引物和探针能够分别在 AAP3 和 COII 基因中检测到三个和六个寄生虫拷贝，并且能够检测到三个拷贝的寄生虫和 。参考试剂盒和我们的方法的灵敏度分别为 98.7%和 98.1%，所有检测中寄生虫基因组的特异性均为 100%。设计的引物和探针在效率和回归系数方面表现良好。对于 AAP3 和 COII 基因，分别为 7 和 0.749 和 0.996 的线性对数范围，对于参考试剂盒使用标准生成曲线，对于寄生虫 DNA 的系列稀释液分别为 0.98 和 0.96。本研究明确检测到 和 ，为其他科学家在多重反应中设计和优化条件 和 开辟了前景。
