收稿日期: 2024-05-10
修回日期: 2024-07-21
网络出版日期: 2024-10-17
基金资助
国家重点研发计划项目(2021YFC2300800);国家重点研发计划项目(2021YFC2300803);国家自然科学基金(31725025)
Establishment and application evaluation of a rapid visualization detection method for Schistosoma japonicum specific nucleic acid fragments based on RPA-CRISPR/Cas12a technology
Received date: 2024-05-10
Revised date: 2024-07-21
Online published: 2024-10-17
Supported by
National Key Research and Development Program of China(2021YFC2300800);National Key Research and Development Program of China(2021YFC2300803);National Natural Science Foundation of China(31725025)
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，建立日本血吸虫特异性核酸片段的快速可视化检测方法，并初步评价其应用价值。 方法 以SjCHGCS20（GenBank：FN356222.1）为靶标序列，设计RPA引物、单链DNA（ssDNA）报告探针和CRISPR RNA（crRNA）序列，优化RPA反应体系中T4重组酶Y、T4重组酶X、T4单链结合蛋白和肌酸激酶的浓度以及RPA反应的温度和时间。建立RPA-CRISPR/Cas12a一管式方法：将优化的RPA反应体系、矿物油和Cas12a切割反应体系放入反应管中，采用优化的反应条件进行RPA扩增后，混合RPA反应产物和Cas12a切割反应体系，Cas12a切割反应后在蓝光割胶仪照射下观察结果，能观察到明亮的绿色荧光则反应产物呈阳性。以100、10-1、10-2、10-3、10-4、10-5和10-6 ng的日本血吸虫成虫基因组DNA为模板进行RPA-CRISPR/Cas12a检测，评价该方法的敏感性；以1 ng日本血吸虫、曼氏血吸虫、卫氏并殖吸虫、华支睾吸虫的基因组DNA为模板进行RPA-CRISPR/Cas12a检测，评价该方法的特异性。取日本血吸虫DNA终浓度为0.2、2、20、200 ng/ml的模拟阳性血清和感染日本血吸虫后第3、7、14、21、28、35和42天小鼠的血清，提取DNA后进行RPA-CRISPR/Cas12a检测，评价该方法的检测效果。 结果 优化的RPA反应体系中，T4重组酶Y、T4重组酶X、T4单链结合蛋白和肌酸激酶的终浓度分别为50、440、200和120 ng/μl；优化的RPA反应条件为40 ℃ 30 min。建立的RPA-CRISPR/Cas12a方法敏感性评价结果显示，当日本血吸虫成虫基因组DNA含量≥ 10-5 ng时反应产物呈阳性；特异性评价结果显示，仅以日本血吸虫成虫基因组DNA为模板的反应产物呈阳性，以曼氏血吸虫、卫氏并殖吸虫、华支睾吸虫基因组DNA为模板的反应产物均呈阴性。日本血吸虫DNA终浓度为2、20、200 ng/ml的模拟阳性血清反应产物呈阳性，终浓度为0.2 ng/ml的模拟阳性血清反应产物呈弱阳性。血吸虫感染后第28和42天的小鼠血清反应产物呈阳性、第35天的呈弱阳性。 结论 本研究建立了检测SjCHGCS20序列的RPA-CRISPR/Cas12a一管式方法，最低检出限为10-5 ng DNA，且与卫氏并殖吸虫、华支睾吸虫、曼氏血吸虫等基因组DNA均无交叉反应，操作简单、反应迅速、特异性高、可借助蓝光激发裸眼观察结果，具有较好的现场检测潜力。
徐国磊 , 冯延叶 , 胡薇 . 基于RPA-CRISPR/Cas12a技术的日本血吸虫特异性核酸片段快速可视化检测方法的建立和应用评估[J]. 中国寄生虫学与寄生虫病杂志, 2024 , 42(5) : 608 -614 . DOI: 10.12140/j.issn.1000-7423.2024.05.007
Objective To develop a rapid visualization method for specific nucleic acid fragments of Schistosoma japonicum using the recombinase polymerase amplification (RPA) technology combined with clustered regularly interspaced short palindromic repeats-associated 12a protein (CRISPR/Cas12a), and to preliminarily evaluate its application value. Methods RPA primers, single-stranded DNA (ssDNA) reporter and CRISPR RNA (crRNA) were designed to target the SjCHGCS20 sequence (GenBank: FN356222.1). The concentrations of T4 recombinase Y, T4 recombinase X, T4 single-stranded binding protein and creatine kinase were optimized in the RPA reaction system, along with the RPA reaction temperature and time. A one-tube RPA-CRISPR/Cas12a method was established by adding the optimized RPA amplification assays, mineral oil and Cas12a reaction system to a reaction tube. After RPA amplification under optimized conditions, the Cas12a reaction was initiated by mixing with the RPA amplification products. The bright green fluorescence signal, indicative of a positive result, was visualized following the CRISPR/Cas12a-mediated cleavage reaction. The sensitivity of the RPA-CRISPR/Cas12a method was evaluated by detecting various amounts of S. japonicum genomic DNA (100, 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 ng). The assay specificity was assessed by detecting genomic DNA (1 ng total) from S. japonicum, S. mansoni, Paragonimus westermani and Clonorchis sinensis. The effectiveness of the method was further evaluated using mice dummy positive sera containing S. japonicum genomic DNA at final concentrations of 0.2, 2, 20 and 200 ng/ml, as well as sera from mice infected with S. japonicum on day 3, 7, 21, 28, 35 and 42 post-infection. Results The optimized RPA reaction system utilized final concentrations of 50 ng/μl for T4 recombinase Y, 440 ng/μl for T4 recombinase X, 200 ng/μl for T4 single-stranded binding protein and 120 ng/μl for creatine kinase, and the optimized reaction condition was 40 ℃ for 30 min. Sensitivity evaluation of established RPA-CRISPR/Cas12a method indicated the reaction products were positive when the S. japonicum genomic DNA content exceeded 10-5 ng. Specificity evaluation indicated the reaction product of S. japonicum genomic DNA was positive, and the reaction products of S. mansoni, P. westermani and C. sinensis genomic DNA were negative. The results of mice dummy positive sera showed that the reaction products of S. japonicum genomic DNA concentrations of 2, 20 and 200 ng/μl were positive, and the reaction product of 0.2 ng/ml was weakly positive. The reaction products of sera from mice infected with S. japonicum on day 28 and 42 post-infection were positive, and the reaction product of sera on day 35 was weakly positive. Conclusion The study successfully established a one-tube RPA-CRISPR/Cas12a method for detecting the SjCHGCS20 sequence. The method has a lowest detectable limit of 10-5 ng for S. japonicum genomic DNA, with no cross-reaction with genomic DNA from S. mansoni, P. westermani or C. sinensis. It is rapid, sensitive, specific and simple to operate, allowing for visual observation of results under blue light, and holds potential for field diagnosis of schistosomiasis japonica.
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