Abstract: Objective　To establish a fluorescent recombinase⁃aided amplification (RAA) assay for detection of Strongyloides stercoralis nucleic acid and to preliminarily evaluate its performance. Methods　Six sets of specific primers targeting S. stercoralis 18S ribosomal RNA (18S rRNA) gene and one fluorescent probe were designed and synthesized. The optimal primer⁃probe set was determined through systematic screening and optimization to establish the fluorescent RAA assay. The assay was evaluated using S. stercoralis genomic DNA at concentrations of 100, 10, and 1 pg/μL, and 100, 10, and 1 fg/μL, as well as recombinant pUC57 plasmids containing the target gene fragments at 1 × 105, 1 × 104, 1 × 103, 1 × 102, 1 × 101, 1 × 100 copies/reaction, to determine the analytical sensitivity. Genomic DNA from Ascaris lumbricoides, Ancylostoma duodenale, Enterobius vermicularis, Angiostrongylus cantonensis, Trichinella spiralis, Clonorchis sinensis, Schistosoma japonicum, and Taenia saginata was used to assess assay specificity. A total of 25 stool samples from patients suspected of S. stercoralis infection were tested by the modified Baermann funnel technique, PCR, and the established fluorescent RAA assay. The sensitivity, specificity, concordance rate and their 95% confidence intervals (CI) of these three techniques were estimated, and agreement between methods was evaluated using the Kappa coefficient.  Results　Exo⁃4 was identified as the optimal primer set screened from the six primer sets, and the best amplification performance was achieved when the final concentrations of the forward and reverse primers were 0.44 μmol/L and a probe concentration was 0.20 μmol/L. The limit of detection of the fluorescent RAA assay was 100 fg/μL for genomic DNA of S. stercoralis and 1 × 100 copies/reaction for recombinant plasmids. Specific fluorescence signals were detected within 5 min, with no cross⁃reactivity observed with A. lumbricoides, A. duodenale, E. vermicularis, A. cantonensis, T. spiralis, C. sinensis, S. japonicum, or T. saginata. Among the 25 clinical stool samples from patients suspected of S. stercoralis infections, the modified Baermann funnel technique and fluorescent RAA assay detected 19 positives and 6 negatives, whereas PCR detected 18 positives and 7 negatives. The fluorescent RAA assay showed a sensitivity of 100.00% [95% CI: (82.35%, 100.00%)], specificity of 100.00% [95% CI: (54.07%, 100.00%)], concordance rate of 100.00% [95% CI: (86.28%, 100.00%)], and a Kappa coefficient of 1.00 [95% CI: (1.00, 1.00)] (P < 0.001) relative to the modified Baermann funnel technique, and a sensitivity of 100.00% [95% CI: (81.47%, 100.00%)], specificity of 85.71% [95% CI: (42.13%, 99.64%)], concordance rate of 96.00% [95% CI: (79.65%, 99.90%)], and a Kappa coefficient of 0.90 [95% CI: (0.70, 1.00)] (P < 0.001). Positive amplification products emitted green fluorescence under a portable blue⁃light device, enabling visual interpretation of results. Conclusions　The fluorescent RAA assay established in this study is rapid, highly sensitive, and highly specific. It enables detection of S. stercoralis nucleic acid under isothermal conditions and allows visual interpretation of results, providing a novel tool for rapid clinical diagnosis and field screening of S. stercoralis infections. 

Key words: Strongyloides stercoralis, Fluorescent recombinase?aided amplification, Nucleic acid detection, 18S rRNA gene, Rapid detection 

摘要： 目的　建立一种基于荧光重组酶介导等温扩增技术（fluorescence recombinase⁃aided amplification，RAA）的粪类圆线虫核酸检测方法，并对其检测性能进行初步评价。方法　以粪类圆线虫18S rRNA基因为靶标，设计并合成6对特异性引物及1条荧光探针，通过系统性筛选与优化，确定最优引物⁃探针体系，用于荧光RAA检测方法的建立。分别以不同浓度粪类圆线虫基因组DNA（100、10、1 pg/μL 和100、10、1 fg/μL）和不同拷贝数的含靶基因片段的pUC57重组质粒（1 × 105、1 × 104、1 × 103、1 × 102、1 × 101、1 × 100 拷贝/反应）为模板进行荧光RAA扩增，确定检测灵敏度。以似蚓蛔线虫、十二指肠钩虫、蛲虫、广州管圆线虫、旋毛虫、华支睾吸虫、日本血吸虫和牛带绦虫基因组DNA为模板进行荧光RAA扩增，评价其检测特异性。收集25份临床疑似粪类圆线虫感染者的粪便样本，分别采用改良贝尔曼漏斗法、PCR法及本研究建立的荧光RAA法进行检测，计算并比较3种检测方法的灵敏度、特异度、符合率及其95%置信区间（confidence interval，CI），采用Kappa一致性系数评价各检测方法之间的一致性。结果　从6对引物中筛选得到最佳引物组合（Exo⁃4），当上、下游引物终浓度为0.44 μmol/L、探针终浓度为0.20 μmol/L时扩增效果最佳。本研究建立的荧光RAA法对粪类圆线虫基因组DNA的最低检测限为100 fg/µL ，对重组质粒的最低检测限为1 × 100 拷贝/反应；该方法在反应5 min内即可检测到特异性荧光信号，且与似蚓蛔线虫、十二指肠钩虫、蛲虫、广州管圆线虫、旋毛虫、华支睾吸虫、日本血吸虫、牛带绦虫均无交叉反应。采用改良贝尔曼漏斗法、PCR法及本研究建立的荧光RAA法对25份疑似粪类圆线虫感染者的临床粪便样本进行检测，结果显示改良贝尔曼漏斗法和荧光RAA法均检出阳性样本19份、阴性样本6份；PCR法检出阳性样本18份、阴性样本7份。相较于改良贝尔曼漏斗法，本研究建立的荧光RAA法灵敏度为100.00%［95% CI：（82.35%，100.00%）］，特异度为100.00%［95% CI：（54.07%，100.00%）］，总符合率为100.00%［95% CI：（86.28%，100.00%）］，Kappa值为1.00［95% CI：（1.00，1.00）］（P < 0.001）；相较于PCR法，本研究建立的荧光RAA法灵敏度为100.00%［95% CI：（81.47%，100.00%）］，特异度为85.71%［95% CI：（42.13%，99.64%）］，符合率为96.00%［95% CI：（79.65%，99.90%）］，Kappa值为 0.90［95% CI：（0.70，1.00）］（P < 0.001）。荧光RAA法扩增阳性样本可在便捷式蓝光电泳仪下呈现明显绿色荧光，实现结果可视化判读。结论　本研究建立的荧光RAA检测方法具有灵敏度高、特异性强、检测快速的特点，可在恒温条件下完成粪类圆线虫核酸检测，并实现结果可视化判读，为粪类圆线虫感染的临床快速诊断及现场筛查提供了新的技术手段。

关键词: 粪类圆线虫, 荧光重组酶介导等温核酸扩增, 核酸检测, 18S rRNA基因, 快速检测