Long-Term Stabilization of Dengue Virus RNA at 37 °C for 14 Months Using Silk Fibroin Films

Diagnosis of dengue virus infections typically relies on RT-PCR-based methods, for which reliable positive controls are essential. Viral RNA is an ideal positive control, but its inherent instability poses a major challenge. Herein, we report a simple and effective method for stabilizing dengue virus RNA by immobilizing it onto silk fibroin films (RNA-SFFs). We evaluated various substrate surfaces for RNA-SFFs preparation and found that the inner surface of sealable bags is optimal for uniform film formation and easy harvesting. Screening different silk fibroin concentrations revealed that even low concentrations (2.8%) effectively preserved RNA well and kept Ct constant for up to 16 days at 25 °C, 37 °C, and even 45 °C (extreme weather for transportations). Due to its rapid film formation and ease of peeling, 7% silk fibroin was selected. Notably, the RNA-SFFs demonstrated robust resistance to UV irradiation, with no significant Ct value changes after 4 h of exposure. Long-term stability testing at -20 °C, 25 °C, and 37 °C showed that dengue serotype 1-4 RNA-SFFs remained stable for the entire duration of the study-up to 56 weeks (approximately 14 months)-at all tested temperatures. These results demonstrate that RNA-SFFs are highly stable, portable, and practical as positive controls for dengue diagnostics, with strong potential for use in on-site and resource-limited settings.