A fast and sensitive real-time PCR assay to detect Legionella pneumophila with the ZEN™ double-quenched probe
Nur Thaqifah Salihah
Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, BE1410
Mohammad Mosharraf Hossain
Institute of Forestry and Environmental Sciences, University of Chittagong, Chittagong 4331,
Minhaz Uddin Ahmed
Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, BE1410
Legionella pneumophila is a waterborne pathogen that causes respiratory ailments including Pontiac fever and Legionnaires’ disease. A culture-free, fast and sensitive detection technique is very important for detection of the pathogen. The present study describes the implementation of rapid cycle real-time PCR in the detection of Legionella pneumophila in water through design and development of a real-time qPCR assay based on the ZENTM probe chemistry. The assay targeted the mip gene for the fast and specific detection of Legionella pneumophila. The novel assay was very specific and fast as the amplification was obtained within 30 minutes. Sensitivity of the assay as evaluated in terms of its limit of detection (LoD) was as low as 100 cells/reaction with the quantification range between 1x103 and 1x107 cells/reaction. The assay has been confirmed for repeatability and reproducibility with approximately less than 1% mean intra- and inter-assay variations (CV%). Therefore, the assay reported can be used for a fast, sensitive and specific culture-free detection and quantification of Legionella pneumophila in water.
