Strepparava Nicole, Wahli Thomas, Segner Helmut, Petrini Orlando
Laboratory of Applied Microbiology, University of Applied Sciences and Arts of Southern Switzerland, Via Mirasole 22a, 6500 Bellinzona, Switzerland.
BMC Microbiol. 2014 Apr 26;14:105. doi: 10.1186/1471-2180-14-105.
Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed.
We describe a qPCR technique based on the single copy gene β' DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes.
This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak.
嗜冷黄杆菌是细菌性冷水病和虹鳟鱼苗综合征的病原体,这两种疾病会导致高死亡率。病原体检测主要通过培养进行,因此需要更快速、灵敏的方法。
我们描述了一种基于单拷贝基因β' 依赖DNA的RNA聚合酶(rpoC)的定量聚合酶链反应(qPCR)技术。其检测限为每个反应20个基因拷贝,定量限为每个反应10³个基因拷贝。对添加已知浓度嗜冷黄杆菌(每个反应10⁶至10¹个细胞)的脾脏进行的测试表明,脾脏组织与引物和探针之间无交叉反应。对无症状和受感染鱼类的水样和脾脏进行筛查表明,在疫情爆发前病原体就已存在,但嗜冷黄杆菌仅在患病鱼类的脾脏中可定量。
这种qPCR可作为一种高度灵敏且特异的方法,用于检测不同样本类型中的嗜冷黄杆菌,无需进行培养。qPCR能够可靠地检测和定量病原体密度低的样本中的嗜冷黄杆菌。嗜冷黄杆菌丰度的定量数据对于调查与感染相关的风险因素以及作为潜在毁灭性疫情爆发前的预警系统可能有用。