目的 结合环介导等温扩增（LAMP）技术与规则成簇的间隔短回文重复序列及相关蛋白12a（CRISPR/Cas12a）系统，建立一种高度敏感、特异的日本血吸虫核酸片段的快速检测方法。方法 选取Sj28S核糖体基因片段作为靶序列，设计LAMP引物、单链DNA（ssDNA）和CRISPR RNA（crRNA），确定各组分浓度，筛选最佳反应体系和条件，建立日本血吸虫核酸LAMP-CRISPR/Cas12a快速荧光检测方法。用不同浓度的日本血吸虫成虫基因组DNA（1 ng/μl、100 pg/μl、10 pg/μl、1 pg/μl、100 fg/μl、10 fg/μl、1 fg/μl）及构建的含Sj28S核糖体基因片段的重组质粒（10⁶、10⁵、10⁴、10³、10²、10¹、10⁰、10^-1拷贝/μl）评估该方法的敏感性，用日本血吸虫、曼氏血吸虫、华支睾吸虫、大片形吸虫、卫氏并殖吸虫、广州管圆线虫、多房棘球绦虫、湄公血吸虫感染的开放拟钉螺以及日本血吸虫感染湖北钉螺和阴性湖北钉螺基因组DNA评价其特异性。构建40、20、10条日本血吸虫尾蚴感染小鼠模型，采集感染后第3~6周小鼠粪样以及感染后第3天及第1~6周小鼠血浆样本，以qPCR法为平行对照，评价所建方法检测日本血吸虫感染鼠粪样与血浆DNA的效果。结果 选择crRNA-2建立LAMP-CRISPR/Cas12a检测方法，筛选ssDNA、crRNA和Cas12a的最适终浓度分别为400、200、200 nmol/L，LAMP预扩增时间为30 min。该方法对日本血吸虫成虫基因组的最低检出限为10 fg/μl，对重组质粒的最低检出限为1拷贝/μl。该方法与曼氏血吸虫、华支睾吸虫、大片形吸虫、卫氏并殖吸虫、广州管圆线虫、多房棘球绦虫等常见寄生虫以及湄公血吸虫感染的开放拟钉螺和阴性湖北钉螺等中间宿主基因组DNA均无交叉反应。LAMP-CRISPR/Cas12a、qPCR法对感染小鼠混合粪样检测阳性率分别为85.42%（41/48）和51.02%（175/343）（χ² = 8.71，P < 0.05），且LAMP-CRISPR/Cas12a法最早可检出小鼠感染后第3周的粪样，早于qPCR法（可检出感染第4周粪样）。LAMP-CRISPR/Cas12a、qPCR法对感染小鼠血浆样本检测阳性率分别为58.3%（28/48）和27.11%（93/343）（χ² = 9.77，P < 0.05）；LAMP-CRISPR/Cas12a和qPCR方法均最早可检出小鼠感染后第3天血浆中的血吸虫循环DNA，阳性率分别为53.06%（26/49）、22.45%（11/49）（χ² = 9.77，P < 0.05）。结论 本研究建立了一种LAMP-CRISPR/Cas12a日本血吸虫核酸快速检测方法，操作简便快捷、高度敏感与特异，且具有较好的早期检测价值，有望为血吸虫病的现场检测提供新的工具。

Objective To develop a highly sensitive and specific assay for rapid detection of Schistosoma japonicum nucleic acid fragments by means of loop-mediated isothermal amplification (LAMP) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) system. Methods The Sj28S ribosomal gene fragment was selected as the target sequence, and LAMP primers, single stranded DNA (ssDNA), and CRISPR RNA (crRNA) were designed. The concentrations of each component was determined, and the optimal reaction system and conditions were screened to establish the LAMP-CRISPR/Cas12a assay for rapid fluorescent detection of S. japonicum nucleic acids. The sensitivity was evaluated using different concentrations of S. japonicum genomic DNA (1 ng/μl,100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl, 1 fg/μl) and recombinant plasmids containing the targeted Sj28S ribosomal gene fragment (10⁶, 10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10^-1 copies/μl), and the specificity was assessed using genomic DNA from S. japonicum, S. mansoni, Clonorchis sinensis, Fasciola gigantica, Paragonimus westermani, Angiostrongylus cantonensis, Echinococcus multilocularis, Neotricula aperta infected with S. mekongi, and Oncomelania hupensis with and without S. japonicum infections. Mice were infected with 40, 20 and 10 cercariae of S. japonicum, and mouse fecal samples were collected 3 to 6 weeks post-infection and plasma samples were collected 3 days and 1 to 6 weeks post-infection. The efficiency of the LAMP-CRISPR/Cas12a assay was examined for detection of stool and plasma DNA samples from mice infected with S. japonicum with qPCR assay as a parallel control. Results The LAMP-CRISPR/Cas12a assay was established based on crRNA-2, and the optimal final concentrations of ssDNA, crRNA, and Cas12a were 400, 200, and 200 nmol/L, respectively, with pre-amplification duration of 30 minutes. The lowest detection limit of this assay was 10 fg/μl for the genome of adult S. japonicum, and 1 copy/μl for the recombinant plasmids, and the assay had no cross reaction with genomic DNA from S. mansoni, C. sinensis, F. gigantica, P. westermani, A. cantonensis, E. multilocularis, N. aperta infected with S. mekongi, and O. hupensis without S. japonicum infections. The positive rates of LAMP-CRISPR/Cas12a and qPCR assays were 85.42% (41/48) and 51.02% (175/343) for detection of the pooled fecal samples from S. japonicum-infected mice, respectively (χ² = 8.71, P < 0.05), and the LAMP-CRISPR/Cas12a assay detected fecal samples from S. japonicum-infected mice as early as 3 weeks post-infection, which was earlier than qPCR assay that detected fecal samples from infected mice 4 weeks post-infection. The positive rates of LAMP-CRISPR/Cas12a and qPCR assays were 58.3% (28/48) and 27.11% (93/343) for detection of plasma samples from S. japonicum-infected mice, respectively (χ² = 9.77, P < 0.05), and both LAMP-CRISPR/Cas12a and qPCR assays detected circulating Schistosoma DNA in the plasma of S. japonicum-infected mice as early as 3 days post-infection, with positive rates of 53.06% (26/49) and 22.45% (11/49), respectively (χ² = 9.77, P < 0.05). Conclusion A simple, rapid and highly sensitive, and specific LAMP-CRISPR/Cas12a assay has been developed for rapid, early detection of S. japonicum nucleic acids, which is a promising new tool for field diagnosis of schistosomiasis.