Detection of Renibacterium salmoninarum in tissue samples by sequence capture and fluorescent PCR based on the 16S rRNA gene

Abstract

The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation were sequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences were found to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotide differences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary to these re-ions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule was constructed to be used as an internal control. The PCR was specific and allowed the detection of DNA equivalent to 1-10 R. salmoninarum genomes per reaction. The DNA preparation with sequence capture and analysis by PCR with a mimic was found to be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in the tissue was reduced, and the relevant DNA was concentrated in the capture step. Furthermore, the use of the mimic molecule in the system assured that false negative results could be identified. (C) 2004 Elsevier B.V. All rights reserved

Keywords

Mimic; PCR; Renibacterium salmoniarum; sequence capture; 16S rRNA

Published in

Veterinary Microbiology
2005, Volume: 105, number: 3-4, pages: 235-243
Publisher: ELSEVIER SCIENCE BV

SLU Authors

• Johansson, Karl-Erik

  • Department of Animal Biosciences, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Animal and Dairy Science
Veterinary Science


Publication identifier

DOI: https://doi.org/10.1016/j.vetmic.2004.11.007

Permanent link to this page (URI)

https://res.slu.se/id/publ/7576