Species-specific polymerase chain reaction primers for Lactococcus garvieae

A dihydropteroate synthase gene from the chromosomal DNA of the fish-pathog enic bacteria Lactococcus garvieae (formerly Enterococcus seriolicida) was cloned. This gene was then chosen as the target for polymerase chain reacti on (PCR). The designated PCR primer set only amplified a 709-bp DNA fragmen t from L. garvieae strains, and did not amplify the same molecular size fra gment from related species of L. lactis, Enterococcus faecalis, E. faecium or beta-haemolytic Streptococcus sp. The kidney tissue of yellowtail, Serio la quinqueradiata (Temminck and Schlegel), a species which is naturally inf ected with L. garvieae, and also kidney tissue samples of healthy yellowtai l were stored in TNES-Urea. The DNA was extracted from tissue samples by a modification of the standard method and by a boiled-extraction method. In p articular, template DNA was utilized within 30 min following extraction and purification by the boiled-extraction method. These species-specific PCR p rimers could amplify a L. garvieae target sequence from yellowtail which we re naturally infected with L. garvieae. The total procedure for the diagnos is of L. garvieae infections in fish, from the point of DNA extraction to o bservation in an agarose gel following electrophoresis, can be performed in less than 4 h.