Author
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Overturf, Kenneth - Ken |
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POWELL, MATT - UNIV OF ID, HAGERMAN |
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Submitted to: Patent Application
Publication Type: Patent Application Publication Acceptance Date: 4/1/2004 Publication Date: 10/1/2004 Citation: Overturf, K.E., Powell, M. 2004. Detection of bkd. Patent Application. Interpretive Summary: Bacterial kidney disease BKD is a systemic infection of salmonids that commonly causes high mortality in populations of wild and propagated salmonids. BKD is caused by Renibacterium salmoninarum. We have developed primers and probes that are specific for the identification and quantification of R. salmoninarum utilizing a real time polymerase chain reaction, RT-PCR, technique. RT-PCR is an existing research technique that relies on precise engineered DNA sequences termed primers for amplification, and a fluorescent tagged binding sequence, or probe, used specifically for the detection and quantification of individual sequences. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye at the level of each individual reaction. Because detection requires not only the amplification of a specific sequence but the binding of the complimentary probe, nonspecific amplification is undetected, hence eliminating certain elements related to the analysis of false positives, a problem with standard PCR. Software analyzes the fluorescence intensity and determines the absolute quantification of the bacterial sequence. This disease is one of the few bacterial diseases that can be spread both horizontally and vertically. Because of BKD's potential to devastate a population, it is common practice to isolate salmon returning to hatcheries, and evaluate them as to their BKD status before utilizing animals for breeding. Currently, BKD analysis is not possible until after the females are milked of their eggs. This results in extra manpower and space in order to remove animals above the culling threshold and keep eggs segregated by female until results are evaluated. A more rapid and sensitive diagnostic method was needed, hence our development of this RT-PCR method. Technical Abstract: Bacterial kidney disease BKD is a systemic infection of salmonids that commonly causes high mortality in populations of wild and propagated salmonids. BKD is caused by Renibacterium salmoninarum. We have developed primers and probes that are specific for the identification and quantification of R. salmoninarum utilizing a real time polymerase chain reaction, RT-PCR, technique. RT-PCR is an existing research technique that relies on precise engineered DNA sequences termed primers for amplification, and a fluorescent tagged binding sequence, or probe, used specifically for the detection and quantification of individual sequences. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye at the level of each individual reaction. Because detection requires not only the amplification of a specific sequence but the binding of the complimentary probe, nonspecific amplification is undetected, hence eliminating certain elements related to the analysis of false positives, a problem with standard PCR. Software analyzes the fluorescence intensity and determines the absolute quantification of the bacterial sequence. This disease is one of the few bacterial diseases that can be spread both horizontally and vertically. Because of BKD's potential to devastate a population, it is common practice to isolate salmon returning to hatcheries, and evaluate them as to their BKD status before utilizing animals for breeding. Currently, BKD analysis is not possible until after the females are milked of their eggs. This results in extra manpower and space in order to remove animals above the culling threshold and keep eggs segregated by female until results are evaluated. A more rapid and sensitive diagnostic method was needed, hence our development of this RT-PCR method. |
