SELEX-based aptamer production: A key step in cholera detection

Vibrio cholerae (V. cholerae) is responsible for the second-highest number of deaths worldwide due to diarrhea. This microorganism is closely associated with cholera outbreaks, both pandemic and epidemic. Therefore, an early rapid detection assay is crucial to prevent the disease's spread. Cholera toxin (Ctx) is a V. cholerae virulence factor that plays a primary and essential role in the pathogenesis of this bacterium. Aptamers, a single-stranded (ss) folded RNA or ssDNA that can bind and detect various nucleic and non-nucleic acid molecules with high affinity and specificity, offer a promising alternative to antibodies in various applications such as biomarker discovery, diagnosis, imaging, and targeted therapy. These aptamers are selected through a process called systematic evolution of ligands by exponential enrichment (SELEX). The present study aimed to produce aptamers by asymmetric PCR, the best conditions obtained for asymmetric PCR included the ratio of 1:80 primers, 20 cycles, and a temperature of 57 °C. After running 11 rounds of SELEX, appropriate aptamers were isolated. The limit of detection (LoD) and dissociation constant (K_D) of the best aptamer for CtxB were calculated for specificity and affinity. The LoD was measured as 100 ± 20 pg/ml, and the K_D was calculated as 320 ± 60 pM.