Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
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
-
File size: 1.3MB
[Abstract] Objective: To establish a recombinase-mediated isothermal amplification (RAA) method that can be used for the detection of Clonorchis sinensis. Methods: The I8S rRNA gene sequence of Clonorchis sinensis was selected as the target sequence, and specific primers and probes were designed, synthesized, and screened to develop a fluorescent RAA assay for rapid detection of Clonorchis sinensis. The sensitivity of the assay was evaluated by performing fluorescent RAA amplification using recombinant plasmids containing DNA fragments with varying copy numbers and genomic DNA from Clonorchis sinensis at different concentrations. The specificity of the assay was assessed by using genomic DNA from Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale, and Schistosoma mansoni as templates. Additionally, the ability of the assay to detect field samples was preliminarily evaluated by extracting DNA from fecal samples containing Clonorchis sinensis eggs and from freshwater fish muscle samples containing metacercariae, followed by fluorescent RAA amplification. Results: A fluorescent RAA assay for the detection of Clonorchis sinensis was successfully established. This assay enables specific amplification of Clonorchis sinensis DNA within 20 minutes at 39°C. Using recombinant plasmids containing DNA fragments with varying copy numbers as templates, the assay achieved a limit of detection as low as 10 copies per reaction. When genomic DNA from Clonorchis sinensis at different concentrations was used as the template, the assay’s limit of detection was as low as 3.7 μL. No positive results were obtained when genomic DNA from Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale, and Schistosoma mansoni was used as the template. The assay successfully detected Clonorchis sinensis infection in human fecal samples, rat fecal samples, and Misgurnus anguillicaudatus samples, demonstrating good performance in detecting field samples. Conclusion: A simple, rapid, sensitive, and specific RAA assay for the detection of Clonorchis sinensis has been successfully established.
Product Inquiry
Contact: qt@qt-bio.com
Product Consultation:+86-510-85385531
Technical support:+86-18921157475
Address: No. 97,Xingye Building B,Linghu Avenue,Xiwu District, Wuxi City
WeChat Official Account
Official Official Account
www.300.cn