Evaluation of Aptamer‐Based Rapid Detection Kit Against Streptococcus agalactiae in Juvenile and Adult Red Hybrid Tilapia Under Control Condition

ABSTRACT Streptococcus agalactiae is one of the most important bacterial pathogens causing streptococcosis in tilapia aquaculture, resulting in substantial economic losses worldwide. Rapid on‐site diagnosis is essential for timely disease intervention; however, available laboratory‐based methods are time‐consuming and require specialised equipment. This study evaluated an aptamer‐based lateral flow assay (LFA) for the rapid detection of S. agalactiae in the body mucus of experimentally infected red hybrid tilapia ( Oreochromis spp.). Fish weighing 50 and 150 g were intraperitoneally challenged with an LD₅₀ dose (approximately 1 × 10 7 CFU/mL) of S. agalactiae . Mucus samples were collected at 2, 4, 8, 12, 16, 20 and 24 h post‐infection and analysed using the LFA, with PCR serving as the reference method. The assay detected S. agalactiae as early as 4 h post‐infection in both size groups, while statistically reliable detection was achieved from 8 h onwards. Diagnostic sensitivity increased from 10% to 100% in 50 g fish and from 30% to 100% in 150 g fish between 4 and 16 h post‐infection. No cross‐reactivity was observed with Streptococcus iniae or Aeromonas hydrophila , indicating promising analytical specificity within this limited evaluation panel. The assay produced visual results within 15 min and exhibited a detection limit of 6.25 μg/mL total mucus protein, whereas PCR detected down to 3.125 μg/mL. No significant difference in diagnostic performance was observed between fish size groups. These findings demonstrated that the aptamer‐based LFA establishes a strong proof‐of‐concept for the early detection of S. agalactiae infection using non‐invasive mucus samples under controlled laboratory conditions. While the assay shows significant potential as a practical diagnostic tool, further field validation is required to fully support routine disease surveillance and management in tilapia aquaculture.