Duodu Samuel, Larsson Pär, Sjödin Andreas, Soto Esteban, Forsman Mats, Colquhoun Duncan J
Section for Bacteriology, Norwegian Veterinary Institute, PO Box 750, Sentrum, 0106 Oslo, Norway.
Dis Aquat Organ. 2012 Nov 19;101(3):225-34. doi: 10.3354/dao02514.
Specific identification and differentiation of the 2 subspecies of the fish pathogen Francisella noatunensis, namely, F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis, remains a major diagnostic challenge. Following whole-genome sequencing and analysis of representatives of all major subclades of the genus Francisella, specific genomic regions were identified for each of the subspecies of this fish pathogen. Two specific real-time quantitative PCR assays, directed at hypothetical genes within these regions were developed. Specificity was confirmed by lack of signal and cross-reactivity with the closest relative, F. philomiragia, and other common bacterial fish pathogens. Both assays, used either as monoplex or multiplex, have a limit of detection of 10 genome equivalents. The quantitative sensitivity of the assays was not affected by the presence of kidney tissues or DNA from Atlantic cod Gadus morhua or tilapia Oreochromis sp.
鱼类病原菌诺氏弗朗西斯菌（Francisella noatunensis）的两个亚种，即诺氏弗朗西斯菌诺氏亚种（F. noatunensis subsp. noatunensis）和诺氏弗朗西斯菌东方亚种（F. noatunensis subsp. orientalis）的特异性鉴定和区分，仍然是一项重大的诊断挑战。在对弗朗西斯菌属所有主要亚分支的代表进行全基因组测序和分析之后，针对这种鱼类病原菌的每个亚种鉴定出了特定的基因组区域。针对这些区域内的假定基因开发了两种特异性实时定量PCR检测方法。通过与最密切相关的皮氏弗朗西斯菌（F. philomiragia）以及其他常见的鱼类细菌性病原菌缺乏信号和交叉反应，证实了检测方法的特异性。这两种检测方法，无论是单重使用还是多重使用，检测限均为10个基因组当量。检测方法的定量灵敏度不受大西洋鳕鱼（Gadus morhua）或罗非鱼（Oreochromis sp.）肾脏组织或DNA的影响。
