title: Filtration recovery of extracellular DNA from environmental water samples.
abstract: qPCR methods are able to analyze DNA from microbes within hours of collecting water samples, providing the promptest notification and public awareness possible when unsafe pathogenic levels are reached. Health risk, however, may be overestimated by the presence of extracellular DNA (eDNA) that is corecovered by the filtration procedure which is the most commonly used method to concentrate target microbes from environmental waters. Using C. parvum 18S rRNA gene fragment as a representative of eDNA, we examined the impact of filters (types and pore sizes) and physiochemical properties of surface water samples on the recovery of spiked DNA. Our results indicated that binding affinities of various filter membranes were quantifiably different for eDNA fragments with the polycarbonate (PC) binding the least and mixed cellulose acetate and cellulose nitrate (MCE) binding the most as evidenced by up to 16% recovery of the spiked plasmid DNA with a pore size of 0.2 μm. Water quality parameters also had a distinct influence on the recovery of eDNA which was enhanced by the presence of high total suspended solid (TSS) concentrations and reduced pH. At pH 5.5, with 150 mg/L of clay, DNA recovery was increased to as much as 18%. By shielding the negative charge, thus increasing the interaction of DNA and colloids, the increase of Na(+) and Ca(2+) concentrations resulted in more DNA binding and consequently more recovery from environmental water samples. Therefore, in addition to analytical uncertainties, potential differences in qPCR data from filtered waters characterized with low pH and high TSS and ionic strength should be considered in pollution assessments.
authors.affiliation: National Research Council, U.S. Environmental Protection Agency, 919 Kerr Research Drive, Ada, Oklahoma 74820, USA.
authors.forename: Zhanbei
authors.initials: Z
authors.lastname: Liang
authors.forename: Ann
authors.initials: A
authors.lastname: Keeley
mesh_terms: D016785:Cryptosporidium parvum / Q000235:genetics; D016054:DNA, Protozoan / Q000302:isolation & purification*; D005374:Filtration; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D009994:Osmolar Concentration; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D060753:Water Quality
chemical_list: D016054:DNA, Protozoan; D008567:Membranes, Artificial; D012337:RNA, Ribosomal, 18S