Rapid RPA ‐Cas12a Assay for Streptococcus agalactiae Serotype III in Fish Featuring Photocontrolled One‐Pot Detection

Streptococcus agalactiae is a primary pathogen responsible for significant mortality in tilapia, a critical and affordable source of animal protein. Among the various serotypes affecting aquaculture, serotype III is increasingly recognized for its high virulence and zoonotic potential, with multidrug resistance reported in some lineages. However, diagnostic tools that combine serotype-level resolution with field-deployability remain scarce. In this study, we developed a rapid and specific CRISPR-based detection platform targeting the cpsH gene of S. agalactiae serotype III. Combining recombinase polymerase amplification and CRISPR detection, the standard two-step RPA-Cas12a assay achieved a limit of detection (LoD) of 100 copies per reaction, with no cross-reactivity observed against closely related cpsH homologues from serotypes Ia, Ib and VI. The entire workflow can be completed within 1 h at a constant temperature of 37°C, providing visual readouts that can be easily and unambiguously interpreted. Furthermore, we demonstrated that the assay can be streamlined into a photocontrolled one-pot format, which reduces the risk of aerosol contamination while maintaining the same sensitivity as the two-step protocol. When evaluated against simulated DNA-spiked samples and field samples, both assay formats exhibited 100% concordance with each other and with the conventional PCR method, highlighting the potential of this platform as a step toward rapid, serotype-specific surveillance in aquaculture.