Establishment of methods for visual and rapid detection of piscine lactococcosis based on isothermal recombinase polymerase amplification.

BACKGROUND

Lactococcosis is a bacterial disease in fish caused by several Lactococcus species, including L. garvieae and L. petauri, with clinical signs frequently overlapping with those caused by other streptococcal species. This diagnostic overlap necessitates a prompt and precise diagnostic tool for guiding accurate treatment and control. To address this diagnostic challenge, we developed basic recombinase polymerase amplification (basic-RPA) and RPA combined with lateral flow dipstick (RPA-LFD) assays for the detection of lactococcosis-associated Lactococcus species.

RESULTS

Both methods exhibited a detection limit of 103 copies/µL for the recombinant plasmid pMD19-adhE-ywdF and 10 pg/µL for L. garvieae genomic DNA. Specificity testing using DNA from L. garvieae, Streptococcus agalactiae, Aeromonas hydrophila, Edwardsiella piscicida, and Streptococcus iniae confirmed that amplification occurred exclusively with L. garvieae, showing specificity against the tested panel. The assay also detected L. petauri, indicating that it recognizes both major lactococcosis-associated Lactococcus species. When tested with the plasmid standard, the RPA assays were 100-fold more sensitive than PCR; with genomic DNA, all three methods showed the same detection limit. When applied to 20 samples from experimentally infected tilapia, the RPA-LFD results were fully consistent with those of conventional PCR.

CONCLUSIONS

The established methods are straightforward, sensitive, and specific, offering a promising candidate tool for the rapid on-site diagnosis of piscine lactococcosis.