Research on the Combination of Isothermal Amplification and Immunochromatography Detection Method of Novel Coronavirus Nucleic Acid

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Objective: To develop a rapid and sensitive detection method for SARS-CoV-2 by combining recombinase polymerase amplification (RPA) with fluorescence immunochromatography. Methods: Specific primers targeting the SARS-CoV-2 nucleocapsid (N) protein gene were designed and modified with fluorescein isothiocyanate (FITC) and biotin. RPA conditions, including primer concentration, magnesium acetate dosage, temperature, and amplification time, were optimized. Immunochromatographic test strips were prepared using FITC-labeled antibodies and streptavidin-coated nitrocellulose membranes. Performance parameters such as sensitivity, specificity, stability, and repeatability were evaluated. Results: By optimizing the RPA reaction conditions and adjusting the immunochromatographic parameters (BY120 nitrocellulose membrane, Type 165 absorbent paper, control line antibody concentration: 0.6mg/mL, detection line streptavidin concentration: 0.8 mg/mL), the system achieved enhanced performance. The immunochromatographic strips achieved a detection limit of 25 pg/mL for SARS-CoV-2, demonstrating 3 fold higher sensitivity than colloidal gold-based strips (100 pg/mL) and a 62-minute reduction in detection time compared to RTqPCR. No cross-reactivity was observed with influenza A/B or respiratory syncytial virus. Stability and repeatability coefficients of variation (CV) were$<10 \%$, confirming robust repeatability. Conclusion: This integrated RPAimmunochromatography method provides a rapid, sensitive, and user-friendly platform for SARS-CoV-2 detection, addressing gaps in existing diagnostic technologies and offering potential for field applications.

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