Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
Development of a Real-Time Fluorescent Reverse Transcription Recombinase-Aided Isothermal Amplification Assay for the Detection of Zika Virus
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【Abstract】 Objective: To establish a real-time fluorescence reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of Zika virus (Zika virus, ZIKV), enabling rapid on-site screening and diagnosis of ZIKV. Methods: Based on genomic sequence alignment, conserved sequences of ZIKV were selected to design specific RT-RAA primers and probes. The sensitivity and reproducibility of the assay were validated using serially diluted ZIKV recombinant plasmids and viral RNA extracts from clinical strains. The specificity of the assay was evaluated by testing it against other arboviruses, including dengue virus, chikungunya virus, and West Nile virus. Results: The established RT-RAA assay can efficiently amplify ZIKV within 30 minutes at a constant temperature of 39°C. The assay demonstrated a 95% detection limit as low as 15 copies per reaction when detecting serially diluted ZIKV recombinant plasmids. The assay showed high specificity, with no cross-reactivity observed against dengue virus, chikungunya virus, or West Nile virus, and exhibited excellent reproducibility. Conclusion: The RT-RAA isothermal amplification method developed in this study for ZIKV detection offers advantages such as rapid reaction time, no requirement for sophisticated equipment, and simple operation, making it well-suited for rapid emergency testing.
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