Development and application of a sensitive droplet digital PCR for the detection of red mark syndrome infection in rainbow trout (Oncorhynchus mykiss)

Red Mark Syndrome (RMS) is a skin disease of uncertain etiology affecting farmed rainbow trout (Oncorhynchus mykiss). It consists of single or multiple skin lesions usually localized on the trunk of fish approaching market size. Studies showed as rickettsia-like organism (RLO), currently referred as Midichloria-like organism (MLO), is supposed to be involved in RMS. Midichloria-like has been identified consistently in RMS-affected fish. Its identification within skin and spleen has been possible through the use of PCR methods, including standard PCR, nested PCR and quantitative PCR (qPCR). Droplet digital PCR (ddPCR) is a relatively novel, sensitive, accurate and absolute quantification technique. Recent studies applied successfully this technique to infectious spleen and kidney necrosis virus (ISKNV) in teleosts, proving its superiority when compared to qPCR. The goal of the present study is to establish a sensitive ddPCR method to rapidly detect and quantify MLO DNA. We explored the feasibility of ddPCR to detect MLO from 40 rainbow trout spleen samples and compared the data with qPCR. The detection limit of the ddPCR was found to be 2.25 copy number, which is lower than the 36 copy number determined for TaqMan real-time PCR (qPCR). This indicated that the sensitivity of the ddPCR assay was one order of magnitude higher than the sensitivity of the qPCR assay. The detection results for fish samples showed that the positive detection rate of ddPCR (65%) was higher than that of qPCR (52.5%). The method used has revealed to be specific to MLO and does not cross-react with other fish bacterial DNA. The ddPCR method established in this study shows superiority for detection in minimal volume samples with low bacterial loads and may be used both as surveillance of possible transmission routes and potential sources.