Objective: To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania.
Methods: Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum.
Results: After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA.
Conclusions: A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.
［摘要］ 目的 建立一种基于荧光重组酶介导等温扩增 (recombinase-aided isothermal amplification, RAA) 技术的检测利 什曼原虫核酸的方法。方法 针对利什曼原虫内转录间隔基因序列1 (ITS1) 基因设计用于 RAA 检测的特异性引物和探 针, 通过引物配对筛选、引物和探针浓度优化, 建立检测利什曼原虫核酸的荧光 RAA 法。分别以构建的含利什曼原虫 ITS1 基因序列的不同拷贝数重组质粒和不同浓度利什曼原虫基因组 DNA 为模板进行 RAA 扩增, 评估其检测灵敏度; 以 田鼠巴贝西虫、刚地弓形虫、间日疟原虫、卵形疟原虫、恶性疟原虫、三日疟原虫、杜氏利什曼原虫、婴儿利什曼原虫等其 他经输血传播的寄生虫基因组 DNA 为模板进行 RAA 扩增, 评价其检测特异度。结果 从 9 对引物组合中筛选出 1 对最 佳引物对后, 引物和探针终浓度经优化分别确定为 0.3 μmol/L 和 0.08 μmol/L。所建立的荧光 RAA 法在 39 °C 等温条件下 20 min 内可完成样本核酸检测。采用建立的 RAA 法对田鼠巴贝西虫、刚地弓形虫、间日疟原虫、卵形疟原虫、恶性疟原 虫、三日疟原虫、杜氏利什曼原虫、婴儿利什曼原虫等 8 种寄生虫基因组 DNA 进行检测, 杜氏利什曼原虫、婴儿利什曼原 虫基因组 DNA 在 5 min 内即出现明显荧光信号, 与田鼠巴贝西虫、刚地弓形虫、间日疟原虫、卵形疟原虫、恶性疟原虫、三 日疟原虫无交叉反应。以含利什曼原虫 ITS1 基因序列的重组质粒为模板, 荧光 RAA 法最低检测限为 10 拷贝/μL; 以利 什曼原虫基因组 DNA 为模板, 荧光 RAA 法最低检测限为 1 fg/μL。结论 成功建立了一种基于荧光 RAA 法的利什曼原 虫核酸检测技术, 该方法反应快速、操作简便、灵敏度和特异度均较高, 可用于利什曼原虫病流行区现场筛查。.
Keywords:
Detection efficiency; Internal transcribed spacer 1;