Use of random DNA amplification to generate specific molecular probes for hybridization tests and PCR-based diagnosis of Yersinia ruckeri

Abstract

We have developed a fast and convenient detection method for the etiological agent of enteric redmouth disease in rainbow trout, the bacterium Yersinia ruckeri, using the random amplification of polymorphic DNA (RAPD) technique to design specific primers for a polymerase chain reaction (PCR)-based diagnosis. In the RAPD genomic fingerprint of Y ruckeri, a specific band was observed which gave no cross-hybridization with the genomes of other bacteria in Southern blot analysis. This band was cloned, sequenced, and found to bear no homology with, known DNA sequences. Two primers were then synthesized to amplify by PCR the fragment lying between the terminal RAPD primer sequences of the band. The PCR assay detected specifically 3 serotypes of V ruckeri (serotypes O1, O2, and unknown) in samples with whole bacteria. It also detected the bacterium in kidney tissue from infected trout after brief digestion with proteinase K. Sample preparation was kept simple to minimize the risk of false positives due to inter-sample contamination Because of its speed, inherent sensitivity, and apparent specificity we concluded that this diagnostic system was preferable to conventional bacteriological diagnostic tests.