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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR ANALYSIS OF VIRULENCE DETERMINANTS OF SELECT BACTERIA IN FISH DISEASES

Location: Aquatic Animal Health Research

Title: Rapid detection of Flavobacterium columnare in fish using TaqMan real-time polymerase chain reaction assay

Authors
item Panangala, Victor
item Shoemaker, Craig
item Klesius, Phillip

Submitted to: American Fisheries Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 30, 2007
Publication Date: June 4, 2007
Citation: Panangala, V.S., Shoemaker, C.A., Klesius, P.H. 2007. Rapid detection of Flavobacterium columnare in fish using TaqMan real-time polymerase chain reaction assay. American Fisheries Society Annual Meeting Proceedings. page 27.

Technical Abstract: Columnaris disease caused by Flavobacterium columnare, a ubiquitous Gram-negative organism prevalent among both warm and cold water reared fish species is important from an economical perspective to the aquaculture industry, worldwide. Because, in commercial aquaculture large numbers of fish are reared in confined space, rapid detection of F. columnare infection is imperative to prevent the spread of disease. We developed a TaqMan-based real-time polymerase chain reaction (PCR) targeting a 113 bp nucleotide region of the chondroitin AC lyase gene of F. columnare G4. Specificity of the assay evaluated with 20 authentic F. columnare isolates and 15 other taxonomically or ecologically related bacteria revealed that the primers and probes were specific for detection of F. columnare. The sensitivity limit of detection of F. columnare in pure cultures, over a range of dilutions (3.1 x 10 to the zero power -3.1 x 10 to the sixth power CFU per mL) was observed to be ~3 bacterial cells. The lowest limit of detection in nucleic acids from pure culture of F. columnare was 5.4 fg and the assay was linear (R square = 0.994) over the range 5.4 ng – 5.4 fg. In experimentally F. columnare-infected fish, blood, gill and kidney tissues yielded 3.4 x 10 to the zero power to 9.5 x 10 to the fifth power CFU per ml by TaqMan real-time PCR. The TaqMan real-time PCR results revealed 100% agreement with the bacteriological culture conducted in parallel on F. columnare spiked tissues and samples from infected fish. The TaqMan real-time PCR is specific and sensitive for rapid detection and quantitation of F. columnare in infected fish.

Last Modified: 7/23/2014
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