Isolation ssDNA aptamers specific for both live and viable but nonculturable state Vibrio vulnificus using whole bacteria-SEILEX technology

Abstract

Vibrio vulnificus is a ubiquitous marine bacterium that may cause rapid and deadly infection, threatening lives of people living around natural bodies of water, especially in coastal regions. However, traditional culture-based methods are time-consuming and unable to detect Viable But Non-Culturable (VBNC) V. vulnificus cells. In this work, we isolated a batch of detection aptamers specifically binding to V. vulnificus in all culture status. With traditional whole bacteria-SELEX (Systematic Evolution of Ligands by EXponential enrichment), flow cytometer analysis and imaging, we identify 18 candidates and validated two of them (V8 and V13) as applicable aptamers. Their truncated sequences also showed comparable performance. The dissociation constant (KD) value of V8 is shown to be as low as 11.22 ± 1.32 nM. Optimal aptamers V8 and V13 are also validated to be effective to detect different Vibrio vulnificus strains under different binding environments using flow cytometry. As for detection parameters, the LOD of the V8 from cytometry is 29.96 CFU mL⁻¹, and the linear range is 10²–5 × 10⁵ CFU mL⁻¹. This is the first case demonstrating that aptamers can detect the existence of VBNC bacteria as well as live bacteria.