目的 基于重组酶聚合酶等温扩增（RPA）技术和簇状规则间隔短回文重复序列及其相关蛋白 12a（CRISPR/Cas12a）系统建立硕大利什曼原虫的检测方法，以提升皮肤利什曼病的诊断效率。方法 以NCBI已公布的硕大利什曼原虫的内转录间隔区1（internal transcribed spacer 1，ITS1）（GenBank登录号为KM052753.1）片段为靶序列，设计RPA引物、CRISPR RNA（crRNA）和单链DNA（ssDNA），通过筛选RPA引物及优化RPA反应温度，建立RPA-CRISPR/Cas12a检测方法。提取硕大利什曼原虫基因组DNA并扩增ITS1序列，扩增产物装载于质粒pUC18以构建重组质粒，重组质粒经梯度稀释为2.3 × 10⁶、2.3 × 10⁵、2.3 × 10⁴、2.3 × 10³、2.3 × 10²、2.3 × 10¹、2.3 × 10⁰、2.3 × 10^-1 拷贝/µl，以此为模板进行RPA-CRISPR/Cas12a及PCR检测，评价该方法检测重组质粒的敏感性。于200 μl犬全血中加入硕大利什曼原虫前鞭毛体（1.8 × 10⁷个/ml），提取基因组DNA，经梯度稀释得到前鞭毛体浓度为1.8 × 10²、1.8 × 10¹、1.8 × 10⁰和1.8 × 10^-1个/ml的基因组DNA，以此为模板进行RPA-CRISPR/Cas12a检测，评价该方法检测虫体敏感性。以硕大利什曼原虫、婴儿利什曼原虫、刚地弓形虫、吉氏巴贝虫、犬新孢子虫、嗜吞噬细胞无浆体等6种病原体基因组DNA为模板进行RPA-CRISPR/Cas12a检测，评价该方法的特异性。以2.3 × 10⁴ 拷贝/μl的重组质粒为模板，分别进行5次RPA-CRISPR/Cas12a检测，每次3个重复，评价该方法的重复性。10份200 μl犬全血中加入硕大利什曼原虫前鞭毛体1.8 × 10⁵个/ml，同时设置阴性对照，使用本研究建立的RPA-CRISPR/Cas12a方法和PCR方法进行检测，比较两种方法的检测效果。结果 选择F7/R7引物组建立RPA-CRISPR/Cas12a方法，优化的反应条件为41 ℃ 25 min。检测重组质粒敏感性评价结果显示，RPA-CRISPR/Cas12a和PCR方法对重组质粒的最低检测限分别为2.3 拷贝/μl和2.3 × 10³ 拷贝/μl；检测虫体敏感性评价结果显示，RPA-CRISPR/Cas12a方法对于虫体的最低检测限为1.8个/ml。特异性评价结果显示，该方法与婴儿利什曼原虫、刚地弓形虫、吉氏巴贝虫、犬新孢子虫、嗜吞噬细胞无浆体等病原体均无交叉反应；重复性评价结果显示，不同批次检测的荧光值稳定且基本一致，相对标准偏差为4.27%，小于5%；RPA-CRISPR/Cas12a和PCR法检测结果显示，两种方法均准确检出10份含硕大利什曼原虫前鞭毛体的犬全血样品，检出率一致，符合率为100%。结论 本研究建立的硕大利什曼原虫RPA-CRISPR/Cas12a检测方法具有快速、灵敏、特异和可重复的检测能力，可为硕大利什曼原虫感染的诊断提供可靠的技术支持。

Objective To develop a rapid and accurate assay for detection of Leishmania major based on the recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system, so as to improve the diagnostic efficiency of cutaneous leishmaniasis. Methdos Based on the internal transcribed spacer 1 (ITS1) gene sequence of L. major (GenBank accession number: KM052753.1) retrieved from NCBI as the target sequence, RPA primers, CRISPR RNA (crRNA), and single-stranded DNA (ssDNA) reporter probe were designed. An RPA-CRISPR/Cas12a assay was developed by screening RPA primers and optimizing the RPA reaction temperature. Genomic DNA was extracted from L. major, and the ITS1 gene sequence was amplified. The amplification product was cloned into the pUC18 plasmid to construct a recombinant plasmid, which was serially diluted to 8 concentrations of 2.3 × 10⁶, 2.3 × 10⁵, 2.3 × 10⁴, 2.3 × 10³, 2.3 × 10², 2.3 × 10¹, 2.3 × 10⁰, and 2.3 × 10⁻¹ copies/μl as templates for RPA-CRISPR/Cas12a assay and PCR assay to evaluate the detection sensitivity of the plasmid. L. major promastigotes were added to 200 μl of canine whole blood samples at a concentration of 1.8 × 10⁷ parasites/ml, and genomic DNA was extracted, and serially diluted into 4 concentrations of 1.8 × 10², 1.8 × 10¹, 1.8 × 10⁰, and 1.8 × 10⁻¹ parasites/ml as templates for RPA-CRISPR/Cas12a assay to evaluate the detection sensitivity of parasites. Genomic DNA from L. major, L. infantum, Toxoplasma gondii, Babesia gibsoni, Neospora caninum, and Anaplasma phagocytophilum were used as templates for the RPA-CRISPR/Cas12a assay to evaluate the detection specificity. Repeatability of the RPA-CRISPR/Cas12a assay was assessed by performing 5 independent experiments with 3 replicates each using the 2.3 × 10⁴ copies/μl recombinant plasmid as the template. To compare the detection performance of the RPA-CRISPR/Cas12a and the PCR assay, L. major promastigotes were added to 10 samples of 200 μl canine whole blood at a concentration of 1.8 × 10⁵ parasites/ml for RPA-CRISPR/Cas12a and PCR assays, with negative controls set simultaneously. Results The RPA-CRISPR/Cas12a method was established using the F7/R7 primer pair, with optimized reaction conditions set as 41 ℃ for 25 minutes. The limits of detection (LOD) of RPA-CRISPR/Cas12a and PCR assays were 2.3 copies/μl and 2.3 × 10³ copies/μl of the recombinant plasmid, and the LOD of the RPA-CRISPR/Cas12a assay was 1.8 promastigotes/ml of parasites; however, this assay showed no cross-reactivity of L. major with L. infantum, T. gondii, B. gibsoni, N. caninum, or A. phagocytophilum. Repeatability testing showed stable and consistent fluorescence intensities across different batches, with a relative standard deviation of 4.27% (< 5%). Both RPA-CRISPR/Cas12a and PCR assays accurately detected all 10 spiked canine whole blood samples containing L. major promastigotes, achieving the same detection rate and a 100% coincidence rate. Conclusion The RPA-CRISPR/Cas12a assay developed in this study is rapid, sensitive, specific, and reproducible for detection of L. major, which may provide reliable technical supports for diagnosis of L. major infections.