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

重组酶介导的斯氏并殖吸虫等温扩增荧光检测方法的建立及检测效果初步评价

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[Abstract] Objective: To establish a rapid nucleic acid detection method for Paragonimus skrjabini based on recombinase-aided isothermal amplification (RAA) technology, and to preliminarily evaluate its detection performance. Methods: Paragonimus skrjabini, Paragonimus westermani, and Metagonimus yokogawai metacercariae were isolated from freshwater crabs, and their genomic DNA was extracted for molecular identification. Primers and probes targeting the cytochrome c oxidase 1 gene (cox1) of P. skrjabini mitochondrial DNA were designed, synthesized, and screened. The established fluorescent RAA assay was validated using genomic DNA samples of P. skrjabini metacercariae collected from Jiyuan City and Yiyang County, Luoyang City, Henan Province. Sensitivity of the assay was assessed by performing fluorescent RAA amplification with serially diluted recombinant plasmids containing the P. skrjabini cox1 gene sequence, as well as with genomic DNA from P. skrjabini metacercariae. Specificity was evaluated by simultaneously detecting genomic DNA from P. westermani, M. yokogawai, Clonorchis sinensis, and Schistosoma japonicum using the developed fluorescent RAA method. Results: Metacercariae of P. skrjabini, P. westermani, and M. yokogawai were successfully isolated from crab samples and identified at the molecular level. Phylogenetic analysis confirmed their high homology with reference strains of Paragonimus available in GenBank. A robust fluorescent RAA assay for P. skrjabini was established, enabling amplification of genomic DNA from P. skrjabini metacercariae collected in Jiyuan City and Yiyang County, Luoyang City, Henan Province, within 5 minutes, with no amplification observed in negative controls. The limit of detection for the fluorescent RAA assay using recombinant plasmids was as low as 10 copies per liter, yielding positive amplification within 5 minutes. When genomic DNA was used as the template, the assay could detect as little as 10 pg/μL of template DNA, with positive amplification occurring within 10–15 minutes. Notably, the fluorescent RAA method yielded negative results when testing genomic DNA from P. westermani, M. yokogawai, S. japonicum, and C. sinensis.

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重组酶介导的日本血吸虫特异性基因片段核酸等温扩增检测方法的建立-江苏血防所

重组酶介导的隐孢子虫属特异性等温核酸扩增方法的建立及评价-江苏血防所

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重组酶介导的日本血吸虫特异性基因片段核酸等温扩增检测方法的建立-江苏血防所

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重组酶介导的隐孢子虫属特异性等温核酸扩增方法的建立及评价-江苏血防所