目的 基于硫代磷酸化dNTP（dNTPαS）增强重组酶聚合酶扩增（RPA）特异性与灵敏度的特性，结合CRISPR/Cas12a的靶核酸识别与信号放大能力，建立一种疟原虫核酸快速分型检测方法（PRCP）。 方法 使用Primer Premier 6设计通用型RPA引物，SnapGene 6.0软件在通用引物区域内针对不同虫种疟原虫设计特异性gRNA。在反应体系中加入dNTPαS，形成S-RPA扩增反应；利用CRISPR/Cas12a对扩增产物进行分型识别和信号放大输出。依次优化PRCP反应体系中dNTPαS浓度、RPA引物浓度、gRNA浓度、Cas12a浓度、反应温度、反应时间、Cas12a最终切割时间等条件。以浓度梯度为以10⁸、10⁷、10⁶、10⁵、10⁴、10³、10²、10¹ 拷贝/µL的恶性疟原虫质粒为模板进行PRCP检测，评估其灵敏度。以乙型肝炎病毒、巴贝虫、布氏锥虫、甲型流感病毒、乙型流感病毒、肺炎支原体、肺炎衣原体作为对照，评估其特异性。通过添加2 g/L血红蛋白、0.1 mmol/L甘油三酯及1 μmol/L胆红素进行PRCP检测，评估其抗干扰能力。利用混合质粒样品检测PRCP体系区分混合感染的能力，并与厚薄血膜涂片法的临床样品（10份疟原虫感染、10份阴性）检测结果进行一致性比较。 结果 筛选3F3R为通用型疟原虫核酸RPA扩增引物，建立了dNTPαS辅助的RPA扩增技术，构建PRCP系统。PRCP体系的优化参数为：dNTPαS最佳占比为70%，最适引物终浓度为0.50 μmol/L（Rate10为676.36），Cas12a终浓度为0.10 μmol/L（Rate10为338.28），gRNA终浓度为0.10 μmol/L（Rate10为718.90）；RPA反应条件为39 ℃（灰度值32 570 ± 5 045）、20 min（灰度值22 513 ± 156）；以Cas12a切割15 min为检测终点（灰度值8 624 ± 359）。系统灵敏度达100 拷贝/μL及以下；与乙型肝炎病毒、巴贝虫、布氏锥虫、甲型流感病毒、乙型流感病毒、肺炎支原体、肺炎衣原体无交叉反应；能抵抗血红蛋白、甘油三酯、胆红素的干扰；具备检测混合感染的能力。与厚薄血膜涂片法相比，PRCP体系检测的符合率为20/20。 结论 本研究成功建立快速、灵敏、特异的PRCP技术，实现了疟原虫基因的快速分型检测，为疟原虫的早期筛查和精准诊疗提供了新方案。

Objective To establish a rapid nucleic acid assay for genotyping for Plasmodium (PRCP) based on phosphorothioate dNTPs (dNTPαS) enhancement of the specificity and sensitivity of recombinase polymerase amplification (RPA), combined with the target nucleic acid recognition and signal amplification capabilities of CRISPR/Cas12a. Methods Universal RPA primers were designed with the software Primer Premier 6, and specific gRNAs targeting different species of Plasmodium were designed within the universal primer region using the software SnapGene 6.0. Then, dNTPαS was added to the reaction system to generate an S-RPA amplification reaction, and CRISPR/Cas12a was employed for typing recognition and signal amplification output of the amplification product. The dNTPαS concentration, RPA primer concentration, gRNA concentration, Cas12a concentration, reaction temperature, reaction time, and final Cas12a cleavage time in the PRCP reaction system were optimized in sequence. PRCP was performed with P. falciparum plasmids at concentrations of 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹ copies/µL as templates to evaluate its sensitivity, and hepatitis B virus, Babesia, Trypanosoma brucei, influenza A virus, influenza B virus, Mycoplasma pneumoniae, and Chlamydia pneumoniae served as controls to evaluate the specificity, and was conducted with addition of 2 g/L hemoglobin, 0.1 mmol/L triglyceride, and 1 μmol/L bilirubin to evaluate its anti-interference ability. In addition, mixed plasmid samples were used to detect the ability of the PRCP system to distinguish mixed infections, and the consistency was compared between detection of mixed plasmid samples and clinical samples (10 samples of Plasmodium infections and 10 negative samples) with thick and thin blood smears. Results A dNTPαS-assisted RPA assay was established based on 3F3R screened as the universal Plasmodium nucleic acid RPA primer to construct a PRCP system. The optimized parameters for the PRCP system included the optimal proportion of dNTPαS as 70%, the optimal final concentration of primers as 0.50 μmol/L (Rate10 as 676.36), the final concentration of Cas12a as 0.10 μmol/L (Rate10 as 338.28), and the final concentration of gRNA as 0.10 μmol/L (Rate10 as 718.90), and the RPA reaction conditions included 39 ℃ (grayscale value of 32 570 ± 5 045) and 20 minutes (grayscale value of 22 513 ± 156), with Cas12a cleavage for 15 minutes as the detection endpoint (grayscale value 8 624 ± 359). The detection sensitivity of the PRCP system was 100 copies/μL or below, and no cross-reactivity was found with hepatitis B virus, Babesia, T. brucei, influenza A virus, influenza B virus, M. pneumoniae, or C. pneumoniae. The PRCP system was found to resist interference from hemoglobin, triglyceride, and bilirubin, and was able to detect mixed infections. Compared with thick and thin blood smears, the PRCP system showed a 20/20 consistency for detection. Conclusion A rapid, sensitive and specific PRCP assay has been successfully established for rapid genotyping of Plasmodium genes, which provides a novel protocol for early screening and precise diagnosis and treatment of Plasmodium.