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

Abstract

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 Y ruckeri (serotypes 01, 0 2 , 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.

A highly sensitive, non‐invasive qPCR‐based strategy for direct quantification of Yersinia ruckeri in fish faeces

“…Consequently, qPCR has been applied to the diagnosis of a wide range of pathogens from various sources including food products (Postollec, Falentin, Pavan, Combrisson & Sohier, 2011), faecal and environmental samples (B elanger, Boissinot, Clairoux, Picard & Bergeron, 2003;Rinttil€ a, Kassinen, Malinen, Krogius & Palva, 2004), and infected plant and animal tissues (Marancik & Wiens, 2013;Osman & Rowhani, 2006). These applications include assays for detection of Y. ruckeri (Argenton et al, 1996;Bastardo, Ravelo & Romalde, 2012), although commercial applicability of these particular methods and their value as a screening tool are limited, as they involve invasive or destructive sampling of putatively infected fish.…”

Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction

“…Although we did not determine the detection limit with PCR, this method was more sensitive than plate culture; more infected fish were identified with the PCR method than by plate culture. Gibello et al (1999) also found rainbow trout that tested positive for Y. ruckeri by PCR but were negative based on the absence of bacterial growth on TSA and MacConkey agar plates; however, Y. ruckeri was isolated on TSA after tissue from these fish was cultured in enrichment broth for 48 h. A previously published PCR method did not detect Yersinia ruckeri in the blood of rainbow trout (Argenton et al 1996). Similar problems detecting other pathogens in blood have been attributed to PCR inhibitors (Iralu et al 1993, Klein et al 1997).…”

“…The greater speed and sensitivity of PCR compared to bacteriological culture of Yersinia ruckeri were discussed by Argenton et al (1996) and Gibello et al (1999). The use of blood for PCR does not require necropsy, and for experimental study of pathogenesis would allow repeated sampling of individual fish.…”

“…However, Argenton et al (1996) failed to detect Y. ruckeri in blood of infected rainbow trout Oncorhynchus mykiss. In the present study, primers described by Gibello et al (1999) were used to detect and identify Y. ruckeri in blood of rainbow trout.…”

“…The polymerase chain reaction (PCR) can also be used to detect Yersinia ruckeri in spleen, liver, kidney, and feces of infected fish (Argenton et al 1996, Gibello et al 1999. However, Argenton et al (1996) failed to detect Y. ruckeri in blood of infected rainbow trout Oncorhynchus mykiss.…”