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Saleh, Mona (2009): Entwicklung von Testsystemen auf der Basis der "Loop Mediated Isothermal Amplification (LAMP)" Methode zum Nachweis von Yersinia ruckeri, dem Erreger der Rotmaulseuche (ERM) und von Renibacterium salmoninarum, dem Erreger der bakteriellen Nierenkrankheit (BKD) der Salmoniden. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Loop-mediated isothermal amplification (LAMP) is a powerful, innovative gene amplification technique which is emerging as an easy to perform and rapid diagnostic tool for detection and identification of microbial diseases. Early and accurate detection is of paramount importance concerning the diagnosis of the highly contagious bacteria Yersinia ruckeri and Renibacterium salmoninarum. An easy to perform diagnostic technique is also required if assays should be carried out in field inquiries. The method provides a single step, reaction tube assay only requiring a temperature-controlled water bath. In the experiments of the presented study, LAMP assays were conducted for Y. ruckeri (the pathogen causing Enteric Redmouth Disease, ERM) and R. salmoninarum (the pathogen causing Bacterial Kidney Disease, BKD). In the case of ERM, the amplified target was a sequence stretch of the gene yruI/yruR encoding the quorum sensing system which controls the expression of virulence genes. In the case of BKD, a sequence stretch of the gene encoding the major soluble antigen protein (p57) in R. salmoninarum was amplified. This protein indicates an active infection because it is the predominant cell surface-associated and secreted protein by the bacterium. The newly established LAMP assays for ERM and BKD enabled amplification of a stretch of each target gene at a temperature of 63°C in less than one hour, with no need of thermal cycling. Assays are carried out with a reaction mix containing four specific primers, the sample and Bst DNA polymerase. Amplification products were detected by visual inspection, agarose gel electrophoresis, and in real-time using a turbidimeter. Assays specificity were demonstrated using DNAs from other related bacteria yielding no amplification product, and by restriction analysis with HphI and EcoRV enzymes producing a specific bands´ pattern of the amplified products. Compared to regular PCR-based detection methods, the developed LAMP assays were consistently faster and ten-fold more sensitive. A safe detection of the specific sequence stretches with high specificity and efficiency was possible using DNA isolated both from bacterial extracts and from clinical fish specimens. These findings showed that LAMP assays are more sensitive than other detection methods such as time consuming culture methods and PCR assays. In conclusion, for the first time LAMP assays developed and optimised to detect Y. ruckeri and R. salmoninarum were introduced as diagnostic tools. In comparison with the performance of already established diagnostic methods, LAMP assays are superior in sensitivity, rapidness, specificity, and cost-efficiency. Both assays are highly appropriate for application in field inquiries to monitor the spread of ERM and BKD.