Validation of a quantitative PCR assay for the detection of 2 Flavobacterium columnare genomovars
Autor: | Matt J. Griffin, Mark L. Lawrence, Gordon D Gibbs, Michael J. Mauel |
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Jazyk: | angličtina |
Rok vydání: | 2020 |
Předmět: |
0303 health sciences
General Veterinary Genotype 030306 microbiology Columnaris disease Biology biology.organism_classification Real-Time Polymerase Chain Reaction Flavobacterium Microbiology Ictaluridae 03 medical and health sciences Fish Diseases Real-time polymerase chain reaction Flavobacteriaceae Infections Flavobacterium columnare Fish Animals Full Scientific Reports 030304 developmental biology |
Zdroj: | J Vet Diagn Invest |
Popis: | 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. |
Databáze: | OpenAIRE |
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