Behets Jonas, Declerck Priscilla, Delaedt Yasmine, Creemers Bart, Ollevier Frans
Laboratory of Aquatic Ecology, Zoological Institute, Katholieke Universiteit Leuven,Charles Deberiotstraat 32, 3000 Leuven, Belgium.
J Microbiol Methods. 2007 Jan;68(1):137-44. doi: 10.1016/j.mimet.2006.07.002. Epub 2006 Aug 17.
This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.
本研究描述了一种针对嗜肺军团菌的特异性Taqman双重实时荧光定量聚合酶链反应（qPCR）的开发与评估，用于在疑似人工水系统中快速、可靠地定量检测这种人类病原体。该qPCR检测方法对所有1 - 15血清型嗜肺军团菌具有100%的特异性，灵敏度为60个基因组单位/升，扩增效率为98%。通过外源性内部阳性对照检测扩增抑制剂，该对照与嗜肺军团菌DNA使用其自身的引物和探针组同时进行扩增。对于添加了已知数量嗜肺军团菌细菌的自来水和冷却循环水，qPCR检测方法的平均回收率分别为93.0%和56.3%。此外，通过使用超净土壤DNA提取试剂盒，我们能够去除冷却水中普遍存在的扩增抑制剂。通过对30份来自淋浴喷头、水龙头、洗眼器、消防喷头和循环回路的水样进行qPCR和传统培养分析，评估了我们的qPCR检测方法的实用价值。总之，所描述的嗜肺军团菌Taqman双重实时检测方法被证明具有特异性、灵敏性和可重复性。这使其成为一种有前景的方法，可补充当前耗时的培养标准方法。
