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Focusing on the research and development and application of isothermal nucleic acid amplification technology
Combining recombinase-mediated isothermal nucleic acid amplification with fluorescent probe-based rapid detection of Schistosoma japonicum gene fragments—Jiangsu Institute of Parasitic Diseases
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【Abstract】 Objective: To establish a rapid detection method for species-specific genomic fragments of Schistosoma japonicum by combining recombinase-mediated isothermal amplification (RAA) with a fluorescent probe approach. Methods: The SjG28 gene fragment of Schistosoma japonicum was used as the target sequence. Primers and a fluorescent probe were designed and synthesized using DNAMAN 70 software to develop a fluorescent RAA reaction system. The sensitivity of the fluorescent RAA assay was evaluated by amplifying recombinant plasmids containing different copy numbers of the SjG28 gene. The specificity of the assay was assessed by performing fluorescent RAA detection using genomic DNA templates from Schistosoma japonicum, Schistosoma mansoni, Echinococcus granulosus, Ancylostoma duodenale, Ascaris lumbricoides, and Clonorchis sinensis. Fluorescent RAA was used to detect DNA extracted from fecal samples of mice infected with 500, 200, and 50 cercariae of Schistosoma japonicum. Additionally, DNA was extracted from 50 snails that were negative but had been co-inoculated with 1, 2, 3, 4, 5, or 10 positive snails, and the DNA was subjected to fluorescent RAA analysis. Results: Amplification by fluorescent RAA using recombinant plasmids containing different copy numbers of the SjG28 gene yielded clear fluorescent signals within 5 minutes; as the copy number decreased, the time required to detect the fluorescent signal increased accordingly. All amplifications at different copy numbers were completed within 10 minutes, and the lowest detectable plasmid concentration was 10 copies per reaction. No fluorescent signals were detected when the fluorescent RAA assay was performed using genomic DNA templates from Schistosoma mansoni, Echinococcus granulosus, Ancylostoma duodenale, Ascaris lumbricoides, and Clonorchis sinensis. DNA samples extracted from fecal materials of mice infected with varying doses of cercariae showed effective amplification by fluorescent RAA, and the detection could be completed within 15 minutes. DNA extracted from 50 snails that were initially negative but contained different numbers of positive snails also yielded effective amplification by fluorescent RAA, and the detection could be completed within 10 minutes. Conclusion: The fluorescent RAA method established in this study for detecting nucleic acid fragments of Schistosoma japonicum is rapid, highly sensitive, and exhibits excellent specificity.
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