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Evaluation of real‐time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms
Author(s) -
Chang C.W.,
Wu Y.C.,
Ming K.W.
Publication year - 2010
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2010.04708.x
Subject(s) - acanthamoeba , biology , microbiology and biotechnology , biofilm , bacteria , genetics
Aims: To assess two real‐time PCR methods (the Riviere and Qvarnstrom assays) for environmental Acanthamoeba . Methods and Results: DNA extracted from Acanthamoeba castellanii taken from water and biofilms of cooling towers was analysed by the Riviere and Qvarnstrom assays. To quantify environmental Acanthamoeba , the calibration curves (DNA quantity vs cell number) were constructed with samples spiked with A. castellanii . The calibration curves for both quantitative PCR assays showed low variation (coefficient of variation of C t ≤ 5·7%) and high linearity ( R 2 ≥ 0·99) over six orders of magnitudes with detection limit of three cells per water sample. DNA quantity determined by Qvarnstrom assay was equivalent between trophozoites and cysts ( P = 0·49), whereas a significant difference was observed with Riviere assay ( P < 0·0001). Riviere assay failed to detect Acanthamoeba in 21% (15/71) of the environmental samples which were positively detected by Qvarnstrom assay, while one sample (1·4%) was shown positive by Riviere assay but negative by Qvarnstrom assay. Moreover, Acanthamoeba counts by Qvarnstrom assay were greater than those by Riviere assay ( P < 0·0001). Conclusions: Qvarnstrom assay performs better than Riviere assay for detection and quantification of Acanthamoeba in anthropogenic water and biofilms. Significance and Impact of the Study: Qvarnstrom assay may significantly contribute to a better knowledge about the distribution and abundance of Acanthamoeba in environments.