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

• Published in: Veterinary microbiology Vol. 105; no. 3; pp. 235 - 243
• Main Authors: Königsson, Malin Heldtander, Ballagi, Andras, Jansson, Eva, Johansson, Karl-Erik
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.02.2005; Elsevier Science
• Subjects: 16S rRNA; Actinomycetales Infections - diagnosis; Actinomycetales Infections - microbiology; Actinomycetales Infections - veterinary; Animals; bacterial kidney disease; Bacteriology; Base Sequence; Biological and medical sciences; diagnostic techniques; disease detection; disease diagnosis; DNA; DNA, Bacterial - chemistry; DNA, Ribosomal - chemistry; False Negative Reactions; fish diseases; Fish Diseases - diagnosis; Fish Diseases - microbiology; Fishes; fluorescence; Fluorescent Antibody Technique - veterinary; Fundamental and applied biological sciences. Psychology; Kidney - microbiology; kidney diseases; microbial detection; Microbiology; Micrococcaceae - classification; Micrococcaceae - genetics; Micrococcaceae - isolation & purification; Mimic; Miscellaneous; molecular mimicry; molecular sequence data; nucleotide sequences; PCR; polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - veterinary; Renibacterium salmoninarum; ribosomal RNA; RNA, Ribosomal, 16S - genetics; Sensitivity and Specificity; sequence analysis; Sequence capture; Species Specificity; tissue analysis; 16S rRNA; Mimic; Sequence capture; PCR; Renibacterium salmoninarum; Polymerase chain reaction; Microbiology; Ribosomal DNA; 16S-RNA; Bacteria; Detection; Veterinary
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2004.11.007

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 regions 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.