Behets Jonas, Declerck Priscilla, Delaedt Yasmine, Verelst Lieve, Ollevier Frans
Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, Charles De Beriotstraat 32, 3000 Leuven, Belgium.
Water Res. 2007 Jan;41(1):118-26. doi: 10.1016/j.watres.2006.10.003. Epub 2006 Nov 13.
A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l(-1) water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless 'low contamination levels' of water samples (<200 N. fowleri cells l(-1)) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.
开发了一种快速准确的双重实时荧光定量聚合酶链反应（qPCR），用于检测和定量水样中的人类致病变形虫福氏耐格里阿米巴。在本研究中，基于Mp2Cl5基因设计了引物和探针，以在单个双重反应中扩增和定量福氏耐格里阿米巴DNA。qPCR检测限（DL）对应于在双重反应中显示出显著荧光的最低DNA量，其至少90%的阳性对照。使用荧光Taqman技术，发现qPCR对福氏耐格里阿米巴具有100%的特异性，检测限为3个福氏耐格里阿米巴细胞当量，PCR效率为99%。在双重qPCR反应中，定量限（QL）为16个福氏耐格里阿米巴细胞当量（相当于320个福氏耐格里阿米巴细胞当量l(-1)水样），对应于可扩增的最低DNA量，变异系数小于25%。为了检测抑制作用，在每个PCR反应中加入外源性内部阳性对照（IPC），以防止假阴性结果。qPCR与最可能数（MPN）培养结果的比较证实，所开发的qPCR非常适合于在实际水样中快速定量检测这种人类病原体。然而，水样的“低污染水平”（<200个福氏耐格里阿米巴细胞l(-1)）仍需要进行培养方法分析。当其他嗜热耐格里阿米巴非常占优势时，MPN培养方法可能会低估福氏耐格里阿米巴的实际数量，因此在解释数据时需要谨慎。福氏耐格里阿米巴qPCR可能是研究嗜热耐格里阿米巴菌株之间进一步竞争现象的有用工具。
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