Evaluation of DNA isolation procedures for detecting and quantifying environmental Legionella by real-time quantitative PCR

Abstract

Six methods, QiAamp DNA Mini Kit (Q), Q with Sepharose 4B gel column (Q/G), Q with low melting point agarose (Q/L), freeze-thaw/phenol-chloroform lysis (FT-PC), FT-PC/G, and FT-PC/L, were evaluated for their ability to isolate DNA of sufficient quality to quantify Legionella using qPCR. Samples of mixing Legionella pneumophila (ATCC33152) and humic acid (HA, 0–126.8 mg/l) were treated by the six methods. Q, Q/G, Q/L, FT-PC/G, and FT-PC/L removed HA from 1.9–126.8 to <1 mg/l determined by A260 with a spectrophotometer. Q obtained the highest DNA yield, followed by Q/G. Dilution (10- to 100-fold) of DNA arising from extraction using Q, Q/G, FT-PC, or FT-PC/G prevented qPCR inhibition. The highest recovery of cells was found in DNA extracted by Q and diluted 100-fold, and followed by Q/G. The applicability of Q and Q/G with dilution was further validated with cooling tower waters. Q or Q/G with 10-fold dilution increased L. pneumophila detection, whereas 100-fold dilution obtained the highest cell concentrations. Similar results were found for Legionella spp. except that both 10- and 100-fold dilutions increased cell concentrations. Thus, Q with 10-fold dilution is suggested to detect and quantify Legionella spp. and detect L. pneumophila. For L. pneumophila-positive samples, 100-fold diluted DNA must be re-analyzed to accurately quantify L. pneumophila.