Development of a Multiplex Polymerase Chain Reaction Assay for Differentiating Three Lactococcus Species Associated With Piscine Lactococcosis

Piscine lactococcosis is an important bacterial disease of farmed fish. The causative agents, Lactococcus garvieae, Lactococcus petauri and Lactococcus formosensis, are closely related, which complicates species-level identification. We developed a conventional multiplex PCR assay targeting species-specific genes identified by comparative genomic analysis. Average nucleotide identity reassignment of 441 publicly available genome assemblies identified 111 L. garvieae, 255 L. petauri and 75 L. formosensis genomes. Species-specific primers and a tuf-based Lactococcus common control were evaluated using in silico polymerase chain reaction (PCR) against target genomes and 10,460 off-target assemblies representing 474 taxa in 12 genera. Experimental specificity was assessed using six target strains and 19 non-target fish pathogens. Distinct amplicons of 195, 333 and 500 bp were produced for L. garvieae, L. petauri and L. formosensis, respectively, together with a 132-bp control amplicon. No cross-amplification was observed. All target species were detected in mixed-DNA samples and spiked kidney and spleen homogenates from two fish species. Analytical detection limits were estimated based on microscopic cell counts of bacterial suspensions before DNA extraction and ranged from 0.956 to 8.55 cell equivalents per reaction. This assay represents a rapid, low-cost method for differentiating lactococcosis-causing Lactococcus species using standard PCR and agarose gel electrophoresis.