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 (0–30mgl−1) or Fe/HA (0/0–3/100mgl−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 (0.3–3mgl−1) or Fe/HA (0.3/10–3/100mgl−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 (≤3mgl−1) or Fe/HA (≤3/100mgl−1).