目的 开发基于PCR结合簇状规则间隔短回文重复序列及相关蛋白Cas12a（CRISPR/Cas12a）快速检测华支睾吸虫囊蚴的方法。 方法 使用内转录间隔区（ITS）引物以华支睾吸虫基因组DNA为模板进行PCR扩增，取PCR扩增产物、Cas12a蛋白、crRNA、单链DNA（ssDNA）报告分子和焦碳酸二乙酯水，选择其中1~5个试剂，分别制备5个CRISPR反应体系，在紫外光下观察反应结果，验证PCR-CRISPR/Cas12a检测系统的可行性。设计5个浓度梯度（100、200、300、400、500 nmol/L）优化ssDNA报告分子浓度；保持crRNA浓度为50 nmol/L，设计5个比例梯度（1.0 : 1、1.5 : 1、2.0 : 1、2.5 : 1、3.0 : 1）优化Cas12a/crRNA比例，荧光定量PCR仪检测荧光信号。将重组质粒梯度稀释为10个不同浓度（10^-2~10⁷拷贝/μl），PCR扩增、CRISPR反应后检测荧光信号，评价检测系统的灵敏度。提取华支睾吸虫囊蚴、东方次睾吸虫囊蚴、猬迭宫绦虫裂头蚴、派氏异尖线虫和亚洲带绦虫节片基因组DNA，PCR扩增、CRISPR反应后检测荧光信号，评价检测系统的特异性。压片镜检华支睾吸虫病流行区捕获的小型淡水鱼鱼肉，分别挑选出华支睾吸虫囊蚴、东方次睾吸虫囊蚴和其他囊蚴（未鉴定出虫种）等3种囊蚴混合感染，华支睾吸虫和东方次睾吸虫囊蚴的混合感染，华支睾吸虫和其他囊蚴的混合感染，东方次睾吸虫和其他囊蚴的混合感染，单华支睾吸虫囊蚴感染，单东方次睾吸虫囊蚴感染，单其他囊蚴感染和无囊蚴感染等8种鱼肉样品组织，提取DNA后进行PCR-CRISPR/Cas12a检测，在紫外光下观察反应结果。使用GraphPad prism 9.0软件对数据进行单因素方差分析，两两比较采用图基多重检验。 结果 完整的PCR-CRISPR/Cas12a检测系统能够在紫外光下产生荧光信号。ssDNA报告分子浓度为400 nmol/L时，反应体系的荧光值为（40 786 ± 1 758） AU，高于300 nmol/L的（32 029 ± 2 651） AU（P < 0.05），与500 nmol/L的（42 698 ± 4 260） AU差异无统计学意义（P > 0.05），选取ssDNA报告分子浓度为400 nmol/L。Cas12a/crRNA比例为2.0 : 1时，反应体系的荧光值为（48 950 ± 3 723） AU，高于1.5 : 1的（37 700 ± 3 887） AU（P < 0.05），与2.5 : 1的（55 630 ± 3 110） AU差异无统计学意义（P > 0.05），选取Cas12a/crRNA比例为2.0 : 1。灵敏度检测结果显示，重组质粒的浓度为10^-1拷贝/μl时，反应体系的荧光值为（39 336 ± 7 231） AU，与阴性对照的（2 216 ± 743） AU差异有统计学意义（P < 0.05）；当浓度降至10^-2拷贝/μl时，荧光值为（5 451 ± 1 957） AU，与阴性对照的差异无统计学意义（P > 0.05）。特异性检测结果显示，ITS引物能同时PCR扩增华支睾吸虫、东方次睾吸虫、猬迭宫绦虫、派氏异尖线虫和亚洲带绦虫等5种基因组DNA；相同扩增产物的CRISPR检测结果显示，仅以华支睾吸虫基因组DNA为模板的反应体系在紫外光下产生了荧光信号。PCR-CRISPR/Cas12a效果评价检测结果显示，8种不同囊蚴感染的鱼肉样品反应体系中，含有华支睾吸虫囊蚴的4管出现荧光信号。 结论 本研究建立了华支睾吸虫囊蚴的PCR-CRISPR/Cas12a检测方法，该方法灵敏度高、特异性好、高度模块化，具有较好的现场应用潜力。

Objective To develop a method for rapid detection of Clonorchis sinensis metacercaria based on the polymerase chain reaction (PCR) technology combined with clustered regularly interspaced short palindromic repeats-associated 12a protein (CRISPR/Cas12a). Methods PCR amplification was performed using internal transcribed spacer (ITS) primers with the C. sinensis genomic DNA as template. The CRISPR reaction system was prepared by mixing PCR amplification product, Cas12a protein, CRISPR-derived RNA (crRNA), and single stranded DNA (ssDNA) reporter molecule, subsequently, the mixture was observed under ultraviolet light to verify the feasibility of the detection system, in which the ssDNA reporting molecule concentration was optimized by designing 5 gradients (100, 200, 300, 400, 500 nmol/L), and the Cas12a/crRNA ratio was optimized by formulating 5 proportional gradients (1.0 : 1, 1.5 : 1, 2.0 : 1, 2.5 : 1, 3.0 : 1) while maintaining crRNA concentration at 50 nmol/L, while the fluorescence signals were detected by using a fluorescence quantitative PCR instrument. The sensitivity of the detection system was evaluated through PCR and CRISPR reaction using the recombinant plasmid diluted to 10 different concentrations (10^-2-10⁷ copies/μl) to examine the reacted fluorescence signal. In addition, the genomic DNA extracted from C. sinensis metacercaria, Metorchis orientalis metacercaria, Spirometra erinaceieuropaei plerocercoid, Anisakis pegreffii and Taenia asiatica used as the template to assess the specificity of the detection system through PCR and CRISPR reaction, judged by checking the fluorescence signal. Samples of small-sized freshwater fish flesh from the clonorchiasis endemic area was examined by microscopy on crushed smears, from them 8 types of mixed infection were separately selected: the samples infected with mixed metacercaria of C. sinensis, M. orientalis and unknown species, metacercaria of C. sinensis and M. orientalis, C. sinensis and unknown species, M. orientalis and unknown species, C. sinensis alone, M. orientalis alone, metacercaria of unknown species alone, and none cercaria infection. These selected sample were used to extract DNA respectively for performing PCR-CRISPR/Cas12a detection, of which the reaction results were observed under ultraviolet light. The data was analyzed by using software GraphPad Prism 9.0 to perform one-way ANOVA. The comparison between groups was analyzed using graph based multiple test. Results The complete PCR-CRISPR/Cas12a detection system was able to generate fluorescence signal under ultraviolet light. When the reported molecular concentration of ssDNA is 400 nmol/L, the average fluorescence value of the reaction system is (40 786 ± 1 758) AU, which is higher than (32 029 ± 2 651) AU at the level of 300 nmol/L (P < 0.05), but no statistically significant difference (P > 0.05) was found when compared with the vlaue of (42 698 ± 4 260) AU at the level of 500 nmol/L. Therefore, the reported molecular concentration of ssDNA was 400 nmol/L. When the Cas12a/crRNA ratio was 2.0 : 1, the average fluorescence value of the reaction system was (48 950 ± 3 723) AU which was higher than (37 700 ± 3 887) AU with the ratio of 1.5 : 1 (P < 0.05) and no statistically significant difference (P > 0.05) compared to (55 630 ± 3 110) AU with the ratio of 2.5 : 1. Thus, the Cas12a/crRNA ratio was selected as 2.0 : 1. When the concentration of the recombinant plasmid was 10^-1 copies/μl, the fluorescence value of the reaction system was (39 336 ± 7 231) AU, which showed a statistically significant difference compared to the negative control (2 216 ± 743) AU (P < 0.05); when the concentration decreased to 10^-2 copies/μl, the average fluorescence value was (5 451 ± 1 957) AU, which had no statistically significant difference with that of the negative control (P > 0.05). The results of PCR showed that ITS primers could simultaneously amplify 5 kinds of genomic DNA of C. sinensis, M. orientalis, S. erinaceieuropaei, A. pegreffii, and T. asiatica. The CRISPR detection results of the same amplification products showed that only the reaction system using the genomic DNA of C. sinensis as a template generated fluorescence signals under ultraviolet light. The results of PCR-CRISPR/Cas12a detection showed that fluorescence signals were observed in four tubes containing C. sinensis metacercariae in the reaction system of 8 types of fish sample infected with the different metacercaria. Conclusion This study established a PCR-CRISPR/Cas12a method for detection of C. sinensis metacercaria, and the method is highly sensitive, specific and modularized, with significant potential for field application.