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Validation of a quantitative PCR assay for the detection of 2 Flavobacterium columnare genomovars.
- Source :
-
Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc [J Vet Diagn Invest] 2020 May; Vol. 32 (3), pp. 356-362. Date of Electronic Publication: 2020 Apr 20. - Publication Year :
- 2020
-
Abstract
- Flavobacterium columnare is the causative agent of columnaris disease in a variety of fish hosts. Using modifications to previously established protocols, a quantitative PCR (qPCR) assay was validated for the detection of 2 predominant F. columnare genomovars. The oligonucleotide primer and probe combination was designed to amplify a 203-bp region of the chondroitin AC lyase gene (GenBank AY912281) of F. columnare . There were no significant differences in amplification between genomovars. Comparable quantities of genomic DNA from 10 F. columnare strains, 5 representatives of each genomovar, produced similar results. Serial dilutions of purified PCR product demonstrated the limit of sensitivity for the assay was ~ 10 copies per reaction. The presence of gill and spleen tissue did not significantly affect the sensitivity of the assay. Comparably, bacterial DNA detected from the liver and kidney was less sensitive than pure bacterial DNA. However, detection from these tissues was within one order of magnitude of other tissues, indicating this reduction may have minimal analytic significance. This validated assay was used to approximate the minimum infectious dose for F. columnare isolate 94-081 in channel catfish and assess bacterial loads in gill and kidney tissues 48 h post-infection.
- Subjects :
- Animals
Fish Diseases microbiology
Flavobacteriaceae Infections diagnosis
Flavobacteriaceae Infections microbiology
Flavobacterium classification
Flavobacterium genetics
Genotype
Real-Time Polymerase Chain Reaction methods
Fish Diseases diagnosis
Flavobacteriaceae Infections veterinary
Flavobacterium isolation & purification
Ictaluridae
Real-Time Polymerase Chain Reaction veterinary
Subjects
Details
- Language :
- English
- ISSN :
- 1943-4936
- Volume :
- 32
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
- Publication Type :
- Academic Journal
- Accession number :
- 32310022
- Full Text :
- https://doi.org/10.1177/1040638720915760