Objective: To establish a multiplex nucleic acid assay for rapid detection of Echinococcus multilocularis, E. granulosus and E. shiquicus based on the recombinase-aided isothermal amplification assay (RAA) and to preliminarily assess its diagnostic efficiency.
Methods: The mitochondrial genomic sequences of E. multilocularis (GenBank accession number: NC_000928), E. granulosus (GenBank accession number: NC_044548) and E. shiquicus (GenBank accession number: NC_009460) were used as target sequences, and three pairs of primers were designed based on the RAA primer design principle and synthesized for the subsequent multiple RAA amplification. The genomic DNA of E. multilocularis, E. granulosus and E. shiquicus at different concentrations and the recombinant plasmids containing the target gene at various concentrations were amplified to evaluate the diagnostic sensitivity of the multiplex RAA assay, and the genomic DNA of E. multilocularis, E. granulosus, E. shiquicus, Taenia multiceps, T. saginata, T. asiatica, Dipylidium caninum, T. hydatigena, Toxocara canis, Fasciola hepatica, T. pisiformis, Mesocestoides lineatus and Cryptosporidiumn canis was detected using the multiplex RAA assay to evaluate its specificity. In addition, the reaction condition of the multiplex RAA assay was optimized, and was then employed to detect the tissues with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples to examine its application values.
Results: The multiplex RAA assay was effective to specifically amplify the mitochondrial gene fragments of E. multilocularis, E. granulosus and E. shiquicus within 40 min at 39 °C, with sequence lengths of 540, 430 bp and 200 bp, respectively. This multiplex RAA assay showed the minimum detection limits of 2.0, 2.5 pg/μL and 3.1 pg/μL for detection of the genomic DNA of E. multilocularis, E. granulosus and E. shiquicus, and presented the minimum detection limit of 200 copies/μL for detection of the recombinant plasmids containing E. multilocularis, E. granulosus and E. shiquicus target genes. This multiplex RAA assay was effective to simultaneously detect single and multiple infections with E. multilocularis, E. granulosus and E. shiquicus, but failed to amplify the genomic DNA of T. multiceps, T. saginata, T. asiatica, D. caninum, T. hydatigena, T. canis, F. hepatica, T. pisiformis, M. lineatus and C. canis. In addition, the optimized multiplex RAA assay was effective to detect all positive samples from the tissue samples with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples, which was fully consistent with the detection of the single PCR assay.
Conclusions: A sensitive and specific multiplex nucleic acid assay for rapid detection of E. multilocularis, E. granulosus and E. shiquicus has been successfully established.
［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、细粒棘球绦虫和石渠棘球绦虫的多重核酸检测方法, 并对其检测效果进行初步评价。方法 分别以多 房棘球绦虫 (GenBank 登录号：NC_000928)、细粒棘球绦虫 (GenBank 登录号：NC_044548) 和石渠棘球绦虫 (GenBank 登录 号：NC_009460) 线粒体基因组序列为靶序列, 依据 RAA 引物设计基本原则设计、合成 3 对引物, 并进行多重 RAA 扩增。分别扩增不同浓度 3 种棘球绦虫基因组 DNA 和不同浓度含 3 种靶基因的重组质粒, 以评价多重 RAA 检测方法的敏感性; 同时采用该方法检测 3 种棘球绦虫及多头带绦虫、牛带绦虫、亚洲带绦虫、犬复孔绦虫、泡状带绦虫、犬弓首蛔虫、肝片形 吸虫、豆状带绦虫、中线绦虫和犬隐孢子虫基因组 DNA, 以评价该方法的特异性。优化建立的多重 RAA 法的条件, 再检 测棘球蚴病病变组织样本、模拟犬粪便样本和现场狐狸粪便样本, 以评价该方法的应用价值。结果 所建立的多重 RAA 检测方法可在 39 °C 时 40 min 内特异性扩增多房棘球绦虫、细粒棘球绦虫和石渠棘球绦虫线粒体基因片段, 长度分 别约为 540、430 bp 和 200 bp。该多重 RAA 法对多房棘球绦虫、细粒棘球绦虫和石渠棘球绦虫基因组 DNA 的最低检测限 为 2.0、2.5 pg/μL 和 3.1 pg/μL, 对含多房棘球绦虫、细粒棘球绦虫和石渠棘球绦虫靶基因的重组质粒的最低检测限均可达 到 200 拷贝/μL; 该多重 RAA 法可以同时检测出多房棘球绦虫、细粒棘球绦虫和石渠棘球绦虫单一感染和多重感染, 对多 头带绦虫、牛带绦虫、亚洲带绦虫、犬复孔绦虫、泡状带绦虫、犬弓首蛔虫、肝片形吸虫、豆状带绦虫、中线绦虫和犬隐孢子 虫无扩增。优化后的多重 RAA 法能够检测出全部棘球蚴病病变组织样本及模拟犬粪样本和现场狐狸粪便样本中的阳 性样品, 且与单一PCR 法检测结果完全一致。结论 成功建立了一种可用于多房棘球绦虫、细粒棘球绦虫和石渠棘球绦虫基因组 DNA 快速检测的多重 RAA 法, 特异性和敏感性均较高。.
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