目的 基于重组酶聚合酶扩增（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）技术，建立快速、便捷、高效检测人粪样中十二指肠钩虫的方法，以及时干预、降低疾病传播风险，提高公共卫生水平。 方法 参考十二指肠钩虫线粒体细胞色素c氧化酶亚基Ⅰ（cox1）序列，设计并筛选RPA引物和CRISPR RNA（crRNA），优化crRNA浓度、ssDNA浓度和其他反应条件，建立RPA-CRISPR/Cas12a荧光检测体系。以10⁵、10⁴、10³、10²、10¹、10⁰、10^-1、10^-2拷贝/µl的十二指肠钩虫阳性质粒为模板，分别采用RPA法和RPA-CRISPR/Cas12a法检测最低检出限，评价该方法的敏感度。分别以十二指肠钩虫（10⁵拷贝/µl）、美洲钩虫（20 ng/µl）、异尖线虫（30 ng/µl）、旋毛形线虫（60 ng/µl）基因组DNA为模板，采用RPA-CRISPR/Cas12a法进行检测，评价该方法的特异性。以Kato-Katz法结合半巢式PCR法结果为标准，检测42份粪样（经Kato-Katz法检测31份为钩虫阳性，11份为钩虫阴性），计算RPA-CRISPR/Cas12a法的灵敏度和特异度，Kappa值比较检测结果的一致性。 结果 建立了基于十二指肠钩虫cox1序列的RPA-CRISPR/Cas12a荧光检测体系，在优化的CRISPR/Cas12a体系中，LbCasl2a核酸酶终浓度为50 nmol/L、crRNA探针终浓度为50 nmol/L、ssDNA报告分子终浓度为100 nmol/L。该检测体系的反应条件为37 ℃ 40 min。RPA的最低检出限为10²拷贝/μl，RPA-CRISPR/Cas12a法的最低检出限为10拷贝/μl，后者敏感度更高，且与美洲钩虫、异尖线虫、旋毛形线虫无交叉反应。与Kato-Katz法结合半巢式PCR法比较，RPA-CRISPR/Cas12a检测的灵敏度为19/19，特异度为91.30%（21/23），一致性较好（Kappa = 0.904 8）。 结论 该研究建立的RPA-CRISPR/Cas12a荧光检测体系具备快速、灵敏、特异的检测能力，为公共卫生监测和钩虫病防控提供了可靠技术支持。

Objective To develop a rapid, convenient, and efficient nucleic acid method based on RPA-CRISPR/Cas12a technology for detecting Ancylostoma duodenale in human stool samples to facilitate timely intervention, reduce the risk of disease transmission, and enhance the level of public health. Methods Based on the conserved sequence of mitochondrial cytochrome c oxidase subunit Ⅰ (cox1) of A. duodenale, RPA primers and CRISPR RNA (crRNAs) were designed and screened, while the concentration of crRNA and ssDNA, along with other reaction conditions, were optimized to establish the RPA-CRISPR/Cas12a fluorescence detection system. The minimum detection limits were determined using A. duodenale plasmid samples as templates, at the concentrations of 10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10⁻¹ and 10⁻² copies/μl, employing both the RPA method and the RPA-CRISPR/Cas12a method. The specificity of the RPA-CRISPR/Cas12a method was determined using genomic DNA templates from A. duodenale (10⁵ copies/μl), Necator americanus (20 ng/µl), Anisakis (30 ng/µl), and Trichinella spiralis (60 ng/µl). To assess the sensitivity and specificity of the RPA-CRISPR/Cas12a method, Kato-Katz combined with semi-nested PCR were used as reference standards, and 42 fecal samples (31 positive and 11 negative for A. duodenale based on the Kato-Katz method) were tested. Sensitivity and specificity values for the RPA-CRISPR/Cas12a method were calculated, and the agreement between detection results was analyzed using Kappa statistics. Results A RPA-CRISPR/Cas12a fluorescence detection system targeting the A. duodenale cox1 gene sequence was developed. In the optimized CRISPR/Cas12a system, the final concentrations of LbCas12a nuclease and crRNA probe were both 50 nmol/L, while the ssDNA reporter concentration was 100 nmol/L. The reaction was performed at 37 ℃ for 40 minutes. The minimum detection limit for the RPA assay was 10⁰ copies/μl, while the RPA-CRISPR/Cas12a system achieved a lower detection limit of 10 copies/μl, demonstrating enhanced sensitivity. The system showed no cross-reactivity with N. americanus, Anisakis, and T. spiralis. When compared with the Kato-Katz combined with semi-nested PCR, the RPA-CRISPR/Cas12a assay exhibited a sensitivity of 100% (19/19), a specificity of 91.30% (21/23), and good consistency (Kappa = 0.9048). Conclusion The RPA-CRISPR/Cas12a fluorescent detection system established in this study possesses rapid, sensitive, and specific detectability, providing reliable technical support for public health surveillance and the prevention and control of hookworm disease.