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.

中文翻译：

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定量PCR检测法可用于检测2株黄杆菌基因突变。

柱状黄杆菌是多种鱼类宿主中柱状病的病原。使用以前建立的协议的修改，定量PCR（qPCR）分析被验证用于检测2个主要的F. columnare基因型。设计寡核苷酸引物和探针的组合以扩增F. columnare的软骨素AC裂解酶基因（GenBank AY912281）的203bp区域。基因型之间的扩增没有显着差异。来自10个F. columnare菌株的可比较数量的基因组DNA，每个基因组的5个代表，产生了相似的结果。纯化的PCR产物的系列稀释液表明该方法的灵敏度极限是每个反应约10个拷贝。g和脾脏组织的存在并未显着影响测定的灵敏度。相对而言 从肝脏和肾脏中检测到的细菌DNA不如纯细菌DNA敏感。但是，从这些组织中检测到的信号量在其他组织的一个数量级之内，这表明这种减少可能具有最小的分析意义。该经过验证的测定法被用于估算channel鱼中柱状分离物94-081的最小感染剂量，并在感染后48小时评估ill和肾组织中的细菌载量。