Usefulness of a TaqMan-based polymerase chain reaction assay for the detection of the fish pathogen Flavobacterium psychrophilum

摘要

Aims: This study developed a new diagnostic method for the bacterium Flavobacterium psychrophilum based on a TaqMan polymerase chain reaction (PCR) assay. Methods and Results: Based on reported and newly designed PCR probes, a rapid procedure, that requires no post-PCR processing, was developed for the detection of F. psychrophilum by measuring the fluorescence produced during PCR amplification. Primers were designed to amplify a 971-bp fragment of the 16S rRNA as the target. When different F. psychrophilum strains and other bacterial species, that are taxonomically and ecologically related, were assayed the fluorogenic test was 100% specific in identifying all of the F. psychrophilum strains. The sensitivity of the assay was found to be 1.1 pg DNA and the assay was linear over a range of 0.1 pg-11.2 ng. With pure cultures of F. psychrophilum, the assay was linear over the range 0.4-4.7 * 10~4 cfu and was able to detect 4.7 cfu per reaction. The analysis was reproducible using either extracted DNA or pure culture. Results using artificially infected fish and diseased fry from natural fish farm outbreaks showed that the assay was useful for diagnosis. Conclusions: The data showed that the assay was as specific, sensitive, reproducible and rapid but less toxic than the PCR assays described and so very useful for the diagnosis of these micro-organisms. Significance and Impact of the Study: This new approach permits a rapid, easy and safe routine laboratory diagnosis of F. psychrophilum.