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

重组酶介导等温核酸扩增技术检测细棘球绦虫方法的建立及初步应用评价_周鸿让CDC

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[Abstract] Objective: To establish a novel, sensitive, and specific recombinase-aided isothermal amplification (RAA) technique capable of rapidly detecting *Echinococcus granulosus* G1. Methods: Primers were designed and synthesized targeting the mitochondrial gene sequence of *E. granulosus* G1 (GenBank No. AF297617). EG1 DNA was used as the template for RAA amplification, with the expected product size of 284 bp. Polymerase chain reaction (PCR) was employed as a parallel control. The sensitivity of the RAA method was evaluated by amplifying genomic DNA at varying dilution levels, as well as gradient-diluted pMD19-T(Simple) plasmids containing different copy numbers of the target gene fragment. Additionally, the specificity of the assay was assessed by testing genomic DNA from parasites including *Echinococcus multilocularis*, *Taenia saginata*, *Taenia asiatica*, *Diphyllobothrium latum*, *Dipylidium caninum*, *Toxocara canis*, *Trichuris vulpis*, *Giardia lamblia*, *Fasciola hepatica*, *Paragonimus westermani*, *Clonorchis sinensis*, and *Opisthorchis viverrini*. After optimization, the RAA method was applied to detect *E. granulosus* G1-infected animal tissue samples (10 specimens), simulated positive canine fecal samples from field conditions (3 specimens), and genuine positive large fecal samples collected in situ (5 specimens), aiming to validate the reliability and practicality of this diagnostic tool. Results: The established RAA method demonstrated specific amplification of the target EgG1 gene fragment within 40 minutes, with a detection limit as low as 10 pg. When using recombinant plasmids as templates, the RAA assay could detect as few as 10^4 plasmid copies. Importantly, no cross-reactivity was observed when testing other parasitic species, including *Echinococcus multilocularis*. Furthermore, both infected animal tissues and simulated field-positive fecal samples yielded positive results with the optimized RAA method, consistent with PCR-based detection outcomes. Conclusion: The developed RAA method targeting *E. granulosus* G1 exhibits simplicity, rapidity, and excellent sensitivity and specificity, making it a promising tool for the identification of *E. granulosus* strains and for the molecular diagnosis of echinococcosis.

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重组酶介导等温扩增技术检测疟原虫方法的建立和评价_邵雷

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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

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重组酶介导等温扩增技术检测疟原虫方法的建立和评价_邵雷