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Filtration recovery of extracellular DNA from environmental water samples
Liang, Z. AND Ann Keeley. Filtration recovery of extracellular DNA from environmental water samples. ENVIRONMENTAL SCIENCE & TECHNOLOGY. American Chemical Society, Washington, DC, 47(16):9324-9331, (2013).
qPCR is a powerful and presently fairly commonly used method of quickly quantifying specific types of microbes from environmental water samples. Many reviews have been written about a standardization of qPCR techniques and the interpretation of resulting data, but these aspects of various sample collection parameters have not been thoroughly investigated. An important component of defining the application of a proposed analytical method should incorporate a characterization of capabilities and limitations of testing procedures including technical properties such as biases, upper or lower detection limits, and calibration curves. Our study describes the recovery behavior of extracellular DNA through filtration under conditions most commonly encountered in field studies, indicating that the filtration recovery of eDNA from environmental waters might be a source of analytical errors in the application of qPCR.
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 co-recovered 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 0.2µm. 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 150mg/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.