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Focusing on the research and development and application of isothermal nucleic acid amplification technology
Development and Evaluation of a Recombinase-Aided Isothermal Nucleic Acid Amplification Method Specific to the Genus Cryptosporidium—Jiangsu Institute of Blood-Related Diseases.pdf
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[Abstract] Objective: To establish a recombinase-mediated isothermal amplification (RAA) method suitable for Cryptosporidium detection and to evaluate its sensitivity and specificity. Methods: A specific I8S rRNA nucleotide sequence of the genus Cryptosporidium was used as the target for detection. Primers and fluorescent detection probes were designed and synthesized using Amplx software, and an optimized RAA fluorescence reaction system was established and validated. The sensitivity of the RAA fluorescence assay was evaluated by using recombinant plasmids containing different copy numbers of the 18S rRNA target sequence, genomic DNA from Cryptosporidium oocysts at varying concentrations, and different amounts of Cryptosporidium oocyst genomic DNA as templates. The specificity of the assay was assessed by using genomic DNA from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella, and Shigella as templates. Results: A successful RAA method for Cryptosporidium detection was established. This method enables efficient amplification of the Cryptosporidium genus-specific I8S rRNA gene fragment within 20 minutes at 39°C. Using recombinant plasmids containing different copy numbers of the I8S rRNA target sequence, genomic DNA from Cryptosporidium oocysts at varying concentrations, and different amounts of Cryptosporidium oocyst genomic DNA as templates, the method achieved detection limits of 10 copies/L, 1 ppL, and 1 oocyst per 50 μL, respectively. No fluorescence signals were detected when genomic DNA from Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella, or Shigella was used as template in the RAA fluorescence assay. Conclusion: A RAA method suitable for detecting Cryptosporidium oocyst DNA has been successfully established. This method is simple to perform, rapid in reaction time, and exhibits both high sensitivity and specificity.
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