Conventional PCR provides Leishmania species characterization with even a small amount of biological material. Species-specific primers have been a widely used alternative; however, nonspecific amplifications are a reality, interfering with PCR efficiency. In endemic areas with multiple etiological agents for leishmaniasis, there is a requirement for higher specificity of primers. This study evaluates 3 pairs of primers described for the identification and characterization of Leishmania infantum. Primers RV1/RV2, LEISH1/LEISH2, and FLC2/RLC2 were used with the DNA of L. infantum, Leishmania amazonensis, and Leishmania braziliensis. An initial temperature curve was performed (52–62 C) to determine the optimal annealing temperature, followed by a dilution curve of Leishmania DNA (500 pg/μl, 50 pg/μl, 5 pg/μl, 500 fg/μl, 50 fg/μl, 5 fg/μl, and 0.5 fg/μl) to be used for analytical sensitivity. RV1/RV2 PCR amplified L. infantum and L. amazonensis at all analyzed temperatures; LEISH1/LEISH2 PCR amplified all 3 species of Leishmania, although at some temperatures L. infantum was specifically amplified, and, finally, FLC2/RLC2 PCR amplified only L. infantum at all temperatures analyzed. In terms of sensitivity, RV1/RV2 PCR detected 1 fg of L. infantum DNA and 100 pg of L. amazonensis DNA; LEISH1/LEISH2 PCR detected 1 fg of L. infantum DNA, 100 fg of L. amazonensis DNA, and 10 fg of L. braziliensis DNA; and FLC2/RLC2 PCR detected 10 fg of L. infantum DNA. Thus, PCR with FLC2/RLC2 primers is best suited for the molecular characterization of L. infantum, especially in areas where there is an incidence of more than 1 Leishmania species, such as South America.

传统的 PCR甚至可以用少量的生物材料来鉴定利什曼原虫的物种。物种特异性引物已成为广泛使用的替代方案；然而，非特异性扩增确实存在，会影响 PCR 效率。在利什曼病多种病原流行地区，对引物的特异性要求较高。本研究评估了用于婴儿利什曼原虫鉴定和表征的 3 对引物。引物 RV1/RV2、LEISH1/LEISH2 和 FLC2/RLC2 与婴儿利什曼原虫、亚马逊利什曼原虫和巴西利什曼原虫的 DNA 一起使用。绘制初始温度曲线 (52–62 C) 以确定最佳退火温度，然后绘制利什曼原虫DNA 稀释曲线（500 pg/μl、50 pg/μl、5 pg/μl、500 fg/μl、50 fg） /μl、5 fg/μl 和 0.5 fg/μl）用于分析灵敏度。RV1/RV2 PCR在所有分析温度下均扩增了婴儿乳杆菌和亚马逊乳杆菌；LEISH1/LEISH2 PCR 扩增了所有 3 种利什曼原虫，尽管在某些温度下婴儿利什曼原虫被特异性扩增，最后，FLC2/RLC2 PCR 在所有分析温度下仅扩增婴儿利什曼原虫。在灵敏度方面，RV1/RV2 PCR检测到1 fg婴儿乳杆菌DNA和100​​ pg亚马逊乳杆菌DNA；LEISH1/LEISH2 PCR 检测到 1 fg婴儿乳杆菌DNA、100 fg亚马逊乳杆菌DNA 和 10 fg巴西乳杆菌DNA；FLC2/RLC2 PCR 检测到 10 fg婴儿乳杆菌DNA。因此，使用 FLC2/RLC2 引物进行 PCR 最适合婴儿利什曼原虫的分子表征，特别是在存在超过 1 种利什曼原虫的地区，例如南美洲。