Innovative Technology

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Focusing on the research and development and application of isothermal nucleic acid amplification technology

重组酶介导的等温核酸扩增技术检测多房棘球绦虫方法的建立及初步应用_周鸿让CDC

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[Abstract] Objective: To establish a rapid nucleic acid detection method for Echinococcus multilocularis based on recombinase-aided isothermal amplification assay (RAA), and to evaluate its performance. Methods: A target sequence derived from the mitochondrial gene of E. multilocularis (GenBank accession number: ABO18440) was used to design and synthesize primers according to the RAA reaction principle. These primers were then employed for RAA amplification. Parallel control experiments were conducted using polymerase chain reaction (PCR). The sensitivity of the RAA method was assessed by amplifying genomic DNA of E. multilocularis at various dilution levels, as well as gradient-diluted pMD19-T(Simple) plasmids containing DNA fragments with different copy numbers. Additionally, the specificity of the assay was evaluated by testing genomic DNA from species such as Echinococcus granulosus (G1 type), Taenia saginata, Taenia asiatica, Multiceps multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Clonorchis sinensis, and Opisthorchis viverrini. After optimizing the experimental conditions, the established RAA method was applied to detect 9 animal tissue samples infected with E. multilocularis, 3 simulated positive canine fecal samples mimicking field conditions, and 2 actual field-positive canine fecal samples, thereby validating the reliability and practicality of the method. Results: The developed RAA method demonstrated specific amplification of the target gene fragment from E. multilocularis within 40 minutes. When using genomic DNA of E. multilocularis as the template, the lowest detectable amount by RAA was 10 pE; when using recombinant plasmids as templates, the method could detect as few as 10 plasmid copies. No amplification was observed when the same RAA protocol was applied to test genomic DNA from E. granulosus (G1 type), Taenia saginata, Taenia asiatica, Multiceps multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Clonorchis sinensis, and Opisthorchis viverrini. Furthermore, the RAA method successfully detected both simulated and field-positive canine fecal samples as well as animal tissue samples infected with E. multilocularis, with results consistent with those obtained by PCR. Conclusion: This study has developed an RAA-based detection method that is not only rapid but also highly sensitive and specific, demonstrating promising potential for species identification of E. multilocularis and genetic diagnosis of echinococcosis.

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再送瘟神 消除血吸虫病适宜技术最新研究进展-江苏血防所

重组酶介导的核酸等温扩增荧...血吸虫感染性钉螺的效能评价_叶钰滢

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再送瘟神 消除血吸虫病适宜技术最新研究进展-江苏血防所

Next page

重组酶介导的核酸等温扩增荧...血吸虫感染性钉螺的效能评价_叶钰滢