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

Development and Preliminary Application of a Recombinase-Aided Isothermal Nucleic Acid Amplification Assay for Detecting Multicystic Echinococcus—Zhou Hongrang, CDC

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[Abstract] Objective: To establish a rapid nucleic acid detection method for Echinococcus multilocularis based on recombinase-mediated isothermal amplification (RAA) assay and to evaluate its detection performance. Methods: The mitochondrial gene sequence of Echinococcus multilocularis (GenBank accession number: ABO18440) was used as the target sequence. Primers were designed and synthesized according to the principle of RAA reaction, and the RAA amplification was performed using these primers. Polymerase chain reaction (PCR) was used as a parallel control. The sensitivity of the RAA method was evaluated by amplifying genomic DNA of Echinococcus multilocularis at different dilution levels and by amplifying pMD19-T(Simple) cloned plasmids containing gene fragments with varying copy numbers at gradient dilutions. The specificity of the RAA method was assessed by testing genomic DNA from species including Echinococcus granulosus (G1 type), Taenia saginata, Taenia asiatica, Multiceps multiceps, Dipylidium caninum, Toxocara canis, Trichuris vulpis, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Clonorchis sinensis, and Opisthorchis viverrini. After optimizing the reaction conditions, the established RAA method was applied to detect 9 tissue samples from animals infected with Echinococcus multilocularis, 3 simulated positive canine fecal samples spiked with Echinococcus multilocularis eggs, and 2 field-positive canine fecal samples to verify the reliability and practicality of the method. Results: The established RAA method could specifically amplify the target gene fragment of Echinococcus multilocularis within 40 minutes. Using genomic DNA of Echinococcus multilocularis as the template, the lowest detection limit of the RAA method was 10 pE; when using recombinant plasmids as templates, the method could detect as few as 10 plasmid copies. No amplification products were detected when the RAA method was applied to genomic DNA from Echinococcus granulosus (G1 type), Taenia saginata, Taenia asiatica, Multiceps multiceps, Dipylidium caninum, Toxocara canis, Trichuris vulpis, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Clonorchis sinensis, and Opisthorchis viverrini. The RAA method established in this study yielded positive results when applied to tissue samples from animals infected with Echinococcus multilocularis, as well as to simulated and field-positive canine fecal samples, and the results were consistent with those obtained by PCR. Conclusion: This study has developed an RAA detection method that is rapid, highly sensitive, and specific. It shows great potential for use in species identification of Echinococcus multilocularis and genetic diagnosis of echinococcosis.

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Recombinase-mediated isothermal amplification of nucleic acids... Evaluation of the efficacy of Oncomelania snails infected with Schistosoma—Ye Yuying

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Sending Away the Plague God: Latest Research Advances in Appropriate Technologies for Eliminating Schistosomiasis—Jiangsu Institute of Schistosomiasis Control