Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
Development and Preliminary Evaluation of a Recombinase-Aided Isothermal Nucleic Acid Amplification Assay for Detecting Echinococcus granulosus—Zhou Hongrang, CDC
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【Abstract】Objective: To establish a novel recombinase-mediated isothermal amplification (RAA) technique that is sensitive, specific, and capable of rapidly detecting the G1 genotype of Echinococcus granulosus. Methods: Primers were designed and synthesized based on the mitochondrial gene sequence of Echinococcus granulosus G1 (E. granulosus G1, EgG1; GenBank No. AF297617), using EG1 DNA as the template for RAA amplification. The expected size of the amplification product was 284 bp. Polymerase chain reaction (PCR) was used as a parallel control. RAA was applied to amplify genomic DNA at different dilution levels and to pMD19-T(Simple) plasmid clones containing varying copy numbers of the target gene fragment at gradient dilutions, in order to evaluate the detection sensitivity of the RAA method. The specificity of the RAA method was assessed by testing genomic DNA from Alveolar echinococcus, Taenia saginata, Taenia asiatica, Taenia solium, Dipylidium caninum, Toxocara canis, Trichuris vulpis, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Clonorchis sinensis, and Opisthorchis viverrini. After method optimization, the RAA assay was applied to detect samples from animal tissues infected with EzG1 (10 samples), simulated positive canine fecal samples collected in the field (3 samples), and positive large-fecal samples collected in the field (5 samples), to validate the reliability and practicality of this detection technique. Results: The established RAA method can specifically amplify the target gene fragment of EgG1 within 40 minutes, with a detection limit as low as 10 pg. When using recombinant plasmids as templates, the lowest detectable copy number by RAA was 10^4. All other parasitic species tested—including Alveolar echinococcus—yielded negative results using the established RAA method. The optimized RAA method yielded positive results when applied to animal tissue samples infected with EzG1 and to positive fecal samples (including simulated field samples), and the results were consistent with those obtained by PCR. Conclusion: The developed RAA method targeting EgG1 exhibits simplicity, rapidity, and high sensitivity and specificity. It holds promise for use in the identification of Echinococcus granulosus genotypes and for the molecular diagnosis of echinococcosis.
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