Objective: To establish a nucleic acid assay for detection of Paragonimus skrjabini based on the recombinase-aided isothermal amplification (RAA) technique, and to preliminarily evaluate its detection efficiency.
Methods: The metacercariae of P. skrjabini, P. westermani and Euparagonimus cenocopiosus were isolated from crabs, and genomic DNA was extracted for molecular characterization. The cytochrome coxidase 1 (cox1) gene sequence of P. skrjabini was selected as the target gene fragment, and the primers and probes were designed, screened and synthesized for RAA assay. The genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province were used as templates for verification of the fluorescent RAA assay. The fluorescent RAA assay was performed to detect different concentrations of plasmids containing target gene fragment and P. skrjabini metacercariae genomic DNA to determine the sensitivity. Fluorescent RAA assay was performed with recombinant plasmids containing P. skrjabini cox1 gene sequences at different concentrations and P. skrjabini genomic DNA as templates to evaluate its sensitivity, and the genomic DNA of P. westermani, E. cenocopiosus, Clonorchis sinensis and Schistosoma japonicum was detected with fluorescent RAA assay to evaluate its specificity.
Results: P. skrjabini, P. westermani and E. cenocopiosus metacercariae were isolated from crabs, respectively. Molecular characterization and phylogenetic analysis confirmed their homology with the genes sequences of standard Paragonimus strains in GenBank. A fluorescent RAA assay was successfully established for nucleic acid detection of P. skrjabini, and the genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province was amplified using the fluorescent RAA assay within 5 min, while the negative control was not amplified. If the recombinant plasmid containing P. skrjabini cox1 gene sequences was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 copies/μL, and positive amplification was observed within 5 min. If genomic DNA was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 pg/μL, and all positive amplifications were found within 5 to 10 min. In addition, the fluorescent RAA assay was tested negative for P. westermani, E. cenocopiosus, C. sinensis and S. japonicum.
Conclusions: A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of P. skrjabini, which has potential values in rapid field detection and species identification in freshwater crabs in areas endemic for P. skrjabini.
［摘要］ 目的 建立一种基于重组酶介导的等温扩增 (recombinase-aided isothermal amplification, RAA) 技术的斯氏并殖 吸虫快速核酸检测方法, 并对其检测效果进行初步评价。方法 从溪蟹样本中分离出斯氏并殖吸虫、卫氏并殖吸虫和三 平正并殖吸虫囊蚴, 提取基因组 DNA 进行分子鉴定。以斯氏并殖吸虫线粒体细胞色素 c 氧化酶亚基 I 基因 (cytochrome coxidase 1, cox1) 基因序列作为靶序列设计、制备、筛选引物及探针。以河南省济源市和洛阳市宜阳县斯氏并殖吸虫囊蚴 基因组 DNA 为模板进行荧光 RAA 检测方法验证。以含斯氏并殖吸虫 cox1 基因序列的不同浓度重组质粒和斯氏并殖吸 虫囊蚴基因组 DNA 为模板进行荧光 RAA 扩增, 评价其检测灵敏度; 应用建立的荧光 RAA 法同时检测卫氏并殖吸虫、三 平正并殖吸虫、华支睾吸虫和日本血吸虫基因组 DNA, 评价其检测特异性。结果 从溪蟹样本中分离出斯氏并殖吸虫、卫氏并殖吸虫和三平正并殖吸虫囊蚴, 经分子鉴定和系统进化分析确认其与 GenBank 中并殖吸虫标准株基因序列具有 同源性。成功建立了斯氏并殖吸虫荧光 RAA 检测方法, 可在 5 min 内扩增到河南省济源市和洛阳市宜阳县采集的斯氏 并殖吸虫囊蚴基因组 DNA, 而阴性对照无扩增。以重组质粒为模板, 荧光 RAA 法最低检出限为 10 拷贝/μL 重组质粒, 且 均在 5 min 内出现阳性扩增; 以基因组 DNA 为模板, 可检测到的最低模板 DNA 浓度为 10 pg/μL, 且均在 10 ~ 15 min 内出 现阳性扩增。荧光 RAA 法检测卫氏并殖吸虫、三平正并殖吸虫、日本血吸虫和华支睾吸虫基因组 DNA 结果均为阴性。结论 成功建立了一种基于荧光 RAA 技术的快速、敏感、特异的斯氏并殖吸虫核酸检测方法, 在斯氏并殖吸虫病流行区 溪蟹现场快速检测与虫种鉴定中具有潜在应用价值。.
Keywords:
Detection efficiency; Nucleic acid detection;