Lactococcus garvieae and Lactococcus petauri are Gram-positive bacteria species causing fish lactococcosis, considered as one of the major threats for rainbow trout (Oncorhynchus mykiss) farming in Europe and worldwide. Therefore, novel analytical methods should be explored to rapidly detect the etiological agent. Droplet digital PCR (ddPCR) is a highly sensitive technique for absolute quantification of nucleic acids. The aim of this study was to develop and validate a ddPCR assay to detect and quantify the presence of L. garvieae/petauri DNA in rainbow trout head kidney, cloacal swabs and in rearing water. L. garvieae/petauri DNA dilutions obtained from pure bacterial cultures and from 89 field samples (tissue, swabs, water) were considered for the ddPCR analysis and parallel quantitative PCR (qPCR) analyses. The detection limit of ddPCR was found to be 1.6 DNA copy number for L. garvieae and 1.7 for L. petauri, showing one order of magnitude higher sensitivity than qPCR. Considering field samples, ddPCR had a 4.49% higher detection capacity for positive cases than qPCR. The ddPCR method validated in this study shows specificity for L. garvieae/petauri, high sensitivity in the detection of low bacterial loads and can be used as surveillance tool both on potentially infected trout and environmental samples, as rearing water or sediment.
Development and validation of a digital droplet PCR method for the detection of Lactococcus garvieae/petauri in water, head kidney and cloacal swabs of infected rainbow trout (Oncorhynchus mykiss)
Saccà E.
;Vesca G.;Beraldo P.;Curcio F.;Volpatti D.
2026-01-01
Abstract
Lactococcus garvieae and Lactococcus petauri are Gram-positive bacteria species causing fish lactococcosis, considered as one of the major threats for rainbow trout (Oncorhynchus mykiss) farming in Europe and worldwide. Therefore, novel analytical methods should be explored to rapidly detect the etiological agent. Droplet digital PCR (ddPCR) is a highly sensitive technique for absolute quantification of nucleic acids. The aim of this study was to develop and validate a ddPCR assay to detect and quantify the presence of L. garvieae/petauri DNA in rainbow trout head kidney, cloacal swabs and in rearing water. L. garvieae/petauri DNA dilutions obtained from pure bacterial cultures and from 89 field samples (tissue, swabs, water) were considered for the ddPCR analysis and parallel quantitative PCR (qPCR) analyses. The detection limit of ddPCR was found to be 1.6 DNA copy number for L. garvieae and 1.7 for L. petauri, showing one order of magnitude higher sensitivity than qPCR. Considering field samples, ddPCR had a 4.49% higher detection capacity for positive cases than qPCR. The ddPCR method validated in this study shows specificity for L. garvieae/petauri, high sensitivity in the detection of low bacterial loads and can be used as surveillance tool both on potentially infected trout and environmental samples, as rearing water or sediment.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
