Objective: To establish a rapid nucleic acid detection technique for identification of Echinococcus multilocularis based on the recombinase aided isothermal amplification assay (RAA) and assess its diagnostic efficiency.
Methods: The mitochondrial gene sequence of E. multilocularis (GenBank accession number: AB018440) was used as a target sequence. The primers were designed according to the RAA reaction principle and synthesized, and RAA was performed using the generated primers. E. multilocularis genomic DNA at various concentrations and the pMD19-T (Simple) vector containing various copies of the target gene fragment were amplified using RAA to evaluate its sensitivity for detection of E. multilocularis, and RAA was em- ployed to detect the genomic DNA of E. granulosus G1 genotype, Taenia saginata, T. asiatica, T. multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis to evaluate its specificity. In addition, the optimized RAA was employed to detect nine tissue specimens of E. granulosus-infected animals, 3 fecal samples from E. granulosus-infected dogs and 2 fecal samples from field infected dogs to examine its reliability and feasibility.
Results: The established RAA was able to detect the specific target gene fragment of E. multilocularis within 40 min. The lowest detect limit of RAA was 10 pg if E. multilocularis genomic DNA served as a template. If the re- combinant plasmid was used as a template, the minimally detectable copy number of RAA was 104. In addition, RAA was nega- tive for the genomic DNA of E. granulosus G1 genotype, T. saginata, T. asiatica, T. multiceps, D. caninum, T. canis, T. trichiura, G. lamblia, F. hepatica, P. westermani, F. gigantica and C. sinensis. The established RAA was positive for detection of the tissue specimens of infected animals, and simulated and field dog stool samples.
Conclusions: A rapid, sensitive and specific RAA is established, which shows promising values in identification of E. multilocularis and gene diagnosis of alveolar echinococcosis.
［摘要］ 目的 基于重组酶介导的等温扩增技术 (Recombinase-aided isothermal amplification assay, RAA) 建立一种快速 检测多房棘球绦虫的核酸检测方法, 并进行检测效果评价。方法 以多房棘球绦虫线粒体基因序列 (GenBank 登录号： AB018440) 作为靶序列, 根据 RAA 反应原理设计并合成引物, 利用该引物进行 RAA 扩增。以聚合酶链反应 (PCR) 作为平 行对照, 应用 RAA 方法扩增不同稀释浓度的多房棘球绦虫基因组 DNA 及梯度稀释的含不同拷贝数目的基因片段的 pMD19-T (Simple) 克隆质粒, 以评价 RAA 检测的敏感性。应用此方法检测细粒棘球绦虫 (G1型)、牛带绦虫、亚洲带绦虫、多头带绦虫、犬复孔绦虫、犬弓首蛔虫、毛首鞭形线虫、蓝氏贾第虫、肝片吸虫、卫氏并殖吸虫、大片吸虫以及华支睾吸虫 基因组 DNA, 以评价其特异性。在对建立的方法进行条件优化后, 检测 9 份感染多房棘球蚴的动物组织样本、3 份模拟现 场多房棘球蚴感染阳性犬粪样本以及 2 份现场阳性犬粪样本, 以验证所建立的 RAA 方法的可靠性和实用性。结果 所 建立的 RAA 法可在 40 min 内特异性扩增多房棘球绦虫目的基因片段。以多房棘球绦虫基因组 DNA 为模板, RAA 法最 低检测量为 10 pg; 以重组质粒为模板, RAA 法最低可检出的质粒拷贝数为 104 个。以细粒棘球绦虫 (G1 型)、牛带绦虫、亚洲带绦虫、多头带绦虫、犬复孔绦虫、犬弓首蛔虫、毛首鞭形线虫、蓝氏贾第虫、肝片吸虫、卫氏并殖吸虫、大片吸虫以及 华支睾吸虫基因组 DNA 为模板, 应用所建立的 RAA 法扩增结果均为阴性。应用本研究建立的 RAA 法检测感染多房棘 球蚴的动物组织样本以及模拟和现场阳性犬粪便样本结果均为阳性, 且与PCR法检测结果一致。结论 本研究建立了 一种反应快捷、敏感性和特异性均较高的 RAA 检测方法, 其在多房棘球绦虫虫种鉴定以及棘球蚴病基因诊断方面展现 出较好应用前景。.
Keywords: Diagnostic performance; Echinococcus multilocularis; Nucleic acid detection; Recombinase-aided isothermal amplification assay (RAA).