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

Establishment and Evaluation of a Recombinase-Aided Isothermal Nucleic Acid Amplification Method Specific for Giardia lamblia—Jiangsu Institute of Blood-Related Diseases

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[Abstract] Objective: To establish a nucleic acid detection method for Giardia lamblia based on recombinase-mediated isothermal amplification (RAA) technology and to evaluate its detection sensitivity and specificity. Methods: The β-giardin gene of Giardia lamblia was selected as the target gene for detection. Specific primers and a fluorescent probe were designed and synthesized to establish a fluorescence-based RAA detection system. Fluorescence RAA amplification was performed using recombinant plasmids containing the B-giardin gene target sequence at different copy numbers and genomic DNA of Giardia lamblia at varying concentrations to assess detection sensitivity. Amplification was also carried out using genomic DNA from Giardia lamblia, Schistosoma japonicum, Clonorchis sinensis, Cryptosporidium parvum, Ascaris lumbricoides, Salmonella, and Shigella as templates to evaluate detection specificity. Results: A fluorescence RAA detection method for Giardia lamblia was successfully established. This method enables rapid and specific amplification of the target gene fragment under isothermal conditions (39°C) within 20 minutes. Using recombinant plasmids and genomic DNA of Giardia lamblia as templates, the detection sensitivity of this method reached 10 copies/μL and 1 pg/μL, respectively. No amplification was observed when genomic DNA from Schistosoma japonicum, Clonorchis sinensis, Cryptosporidium parvum, Ascaris lumbricoides, Salmonella, and Shigella was used as templates, demonstrating good specificity. Conclusion: A simple, sensitive, and specific fluorescence RAA method has been established for the detection of Giardia lamblia nucleic acids.

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Development of a Recombinase-Aided Isothermal Amplification Assay for the Detection of Schistosoma japonicum-Specific Gene Fragments—Jiangsu Institute of Parasitic Diseases

Establishment and Preliminary Evaluation of a Recombinase-Aided Isothermal Amplification Method for Species-Specific Nucleic Acid Detection of Clonorchis sinensis—Jiangsu Institute of Blood-Related Diseases

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Development of a Recombinase-Aided Isothermal Amplification Assay for the Detection of Schistosoma japonicum-Specific Gene Fragments—Jiangsu Institute of Parasitic Diseases

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Establishment and Preliminary Evaluation of a Recombinase-Aided Isothermal Amplification Method for Species-Specific Nucleic Acid Detection of Clonorchis sinensis—Jiangsu Institute of Blood-Related Diseases