title: Quantification of Legionella pneumophila by real-time quantitative PCR from samples with humic acid and ferric ion.
abstract: This study determined and overcame the influences of humic acid (HA) and/or ferric ion (Fe) on quantification of Legionella pneumophila by real-time quantitative PCR (qPCR). Four commonly used DNA isolation methods, QiAamp DNA Mini Kit (Q), Q with Sepharose 4B gel column (Q/G), freeze-thaw/phenol-chloroform lysis (FT-PC), and FT-PC/G, were adopted to isolate L. pneumophila DNA from samples containing Fe alone (0-30 mg l(-1)) or Fe/HA (0/0-3/100 mg l(-1)). Among the four DNA isolation methods, Q removed HA effectively and obtained the greatest DNA yield regardless of Fe and HA concentration (P<0.05). For samples containing Fe (0.3-3 mg l(-1)) or Fe/HA (0.3/10-3/100 mg l(-1)), qPCR inhibition was found in all isolated DNA, especially in those obtained by Q/G and FT-PC/G. DNA dilution at either 10 or 100 folds reduced qPCR inhibition and increased cell recovery (P<0.05). Under 10-fold dilution, Q acquired the highest concentrations of L. pneumophila determined by qPCR. Consequently, Q with post 10-fold dilution is suggested prior to qPCR for quantifying L. pneumophila from water containing Fe (≤ 3 mg l(-1)) or Fe/HA (≤ 3/100 mg l(-1)).
authors: Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei City 100, Taiwan.
authors: Nai-Tzu
authors: NT
authors: Chen
authors: Ching-Wen
authors: CW
authors: Chang
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification*; D006812:Humic Substances / Q000032:analysis; D007501:Iron / Q000032:analysis; D016952:Legionella pneumophila / Q000235:genetics*; D016014:Linear Models; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D006812:Humic Substances; D007501:Iron