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

The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation weresequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences werefound to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotidedifferences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary tothese regions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule wasconstructed 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 foundto be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in thetissue was reduced, and the relevant DNAwas concentrated in the capture step. Furthermore, the use of the mimic molecule inthe system assured that false negative results could be identified.

Ballagi, AndrasUppsala universitet,Institutionen för ytbioteknik med Centrum för ytbioteknik,Institutionen för fysikalisk och analytisk kemi(Swepub:uu)aba06160 (författare)
Jansson, Eva (författare)
Johansson, Karl-Erik (författare)
Uppsala universitetInstitutionen för ytbioteknik med Centrum för ytbioteknik (creator_code:org_t)

Av författaren/redakt...: Heldtander König ...; Ballagi, Andras; Jansson, Eva; Johansson, Karl- ...

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