Article pubs.acs.org/est
Filtration Recovery of Extracellular DNA from Environmental Water Samples Zhanbei Liang† and Ann Keeley*,‡ †
National Research Council, and ‡National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 919 Kerr Research Drive, Ada, Oklahoma 74820, United States S Supporting Information *
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 Ca2+ 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.
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INTRODUCTION The microbial contamination of water continues to pose a serious threat to public health in spite of recent efforts to improve water quality.1 In 2010, water quality impairments along the marine coastline and Great Lakes in the United States resulted in more than 24,000 recorded beach closings and water quality advisories primarily due to fecal contaminants.2 Microbial contamination is also the leading cause of river and streamwater deterioration in the United States (USEPA, 2012). Developing mitigation strategies to address fecal pollution of water has been described as “the challenge of the 21st century”,3 and the identification and reduction of fecal pollution sources have been recognized as top priorities for water management practices.4 Culture-based methods of fecal indicator bacteria (FIB) routinely used in water quality assessments are relatively slow, requiring 18 to 96 h to obtain results. These classical methods usually require time-consuming lab work to enrich, cultivate isolates of interest, and provide subsequent systematic differentiation. In addition, injured or stressed microbial cells that are in a viable but nonculturable (VBNC) status usually grow poorly on artificial media further limiting the use of culturebased methods. By directly targeting genetic material, thus reducing measurement times to as little as 2 h,5 quantitative polymerase chain reaction (qPCR) has recently received widespread attention from both researchers and regulators This article not subject to U.S. Copyright. Published 2013 by the American Chemical Society
since the technology has been found to correlate well with health risks.6−8 qPCR methods have also been used in the detection of DNA targets in wastewater effluent, stormwater discharge, and riverine waters with variable levels of success as part of regional water quality management plans or enhanced coastal water quality monitoring programs.9,10 As a result, the USEPA is considering incorporating qPCR techniques into the promulgation of new national water quality criteria. The application of qPCR in water quality monitoring consists of a series of steps including, but not limited to, environmental water collection, sample filtration, DNA extraction, enhancing sample processing efficiency, reducing amplification inhibition, identifying source of extracellular DNA (eDNA), and transforming a DNA signal to cell equivalents (CE). While all steps are critical for the success of qPCR based monitoring procedures, and deserve scrupulous validation and performance evaluation, the focus of this present study is on the filtration step. Filtration is currently the most commonly used method to concentrate bacteria from environmental waters prior to qPCR detection; however, this critical step could potentially recover Received: Revised: Accepted: Published: 9324
April 1, 2013 July 16, 2013 July 19, 2013 July 19, 2013 dx.doi.org/10.1021/es401342b | Environ. Sci. Technol. 2013, 47, 9324−9331
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was cloned using TOPO TA cloning kit (Invitrogen, Carsland, CA) according to the protocol recommended by the manufacturer. Single positive isolate as confirmed by PCR from a freshly streaked plate was inoculated into LB broth containing ampicillin (50 μg/mL) and incubated overnight with shaking. Plasmid was then extracted using the Qiagen Plasmid Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. The extracted supercoiled plasmid DNA was linearized by restriction enzymatic digestion using the enzyme Eco53kI (New England Biolabs, Inc., Beverly, MA) following the recommended procedure. Digested DNA was purified by a QIAquick PCR Purification Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The integrity of the plasmid DNA was then checked by agarose gel electrophoresis using 1-kb DNA ladder as a marker. Concentration of the purified linearized plasmid was estimated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). Copy numbers of plasmid were calculated with the following equation: copies per nanogram = (n × mw) /(NL × 10−9), where n is the length of the plasmid in base pairs, mw is the molecular weight per bp, and NL is the Avogadro constant (6.02 × 1023 molecules per mol). Plasmid DNA was diluted in nuclease free water to a final concentration of 1E8 copies/μL, divided into 200 μL aliquots, and stored at −20 °C until use. Organic and Inorganic Particles. Fine-sized fraction (90%, as determined from the slope of standard curve, and R2 value of >0.95 were verified for all standard curves. Copy numbers of target in the measured samples were determined from the standard curve automatically by SDS software. Effect of Membrane Properties. Plasmid and E. coli suspended in DI water was filtrated through membrane filters of different types and pore sizes to investigate the effect of membrane properties on the recovery of extracellular DNA. Three concentrations of plasmid, 5 × 105, 5 × 106, and 5 × 107 copies/mL, were evaluated. Prepacked sterile membrane filters (diameter 47 mm) tested, which are frequently used for standard microbiological examinations of water quality, included Durapore* polyvinylidene fluoride filter (PVDF, pore size: 0.1 μm, 0.2 and 0.45 μm, Millipore), Supor 200 polyethersulfone filter (PES, 0.2 and 0.45 μm, Pall), Nuclepore Polycarbonate filter (PC, 0.2 and 0.45 μm, Whatman), and mixed cellulose esters filter (MCE, 0.2 and 0.45 μm, Fisher). Filtration and DNA extraction were conducted as described above. An aliquot of plasmid stock solution containing the same copies of plasmid added directly into the 2 mL PowerBead tube was subjected to DNA extraction and used as method control. Effect of Total Suspended Solids (TSS). Influences of the organic and inorganic fractions of TSS on the recovery of eDNA were studied using clay and organic particles as prepared above. For each fraction, particles were added into DI water to obtain a series of suspensions with desired concentrations (1 mg/L to 3log10 mg/L). After spiking plasmid and E. coli cells, the suspension was thoroughly mixed, filtrated through Nuclepore* Polycarbonate membrane filters (0.2 μm pore size) as described above, and proceeded for DNA extraction.
For each concentration level, the amount of TSS in 100 mL of suspension was calculated. An equivalent amount of corresponding fraction together with 5 × 108 copies of plasmid was added into the 2 mL PowerBead tube and subjected to DNA extraction as a method control. Effect of Ionic Composition. A series of CaCl2 or NaCl solutions (concentration ranging from 0.05 mM to 100 mM) were used to test the effect of ionic composition on the filtration recovery of eDNA. An aliquot of each concentration was spiked with clay to a final concentration of 150 mg/L. An aliquot with no clay was also used in this study. The pH value of each sample was adjusted to 7.5 using 0.01 N NaOH. Plasmids and E. coli cells were then spiked into each suspension, mixed, and subjected to filtration through 0.2 μm Nuclepore* Polycarbonate membrane filter and subsequent DNA extraction. Plasmids (5 × 108 copies) and 15 mg of clay added into the PowerBead tube was subjected to DNA extraction and used as method control for the clay spiking serial suspensions. Aliquots of 5 × 108 copies of plasmid processed the same way were used as method control for the nonclay spiking serial solution. Effect of pH. To study the influence of pH on the filtration recovery of eDNA, four types of solutions were used: double distilled water (A), 5 mM NaCl solution (B), DI water suspended with 150 mg/L of clay (C), and 5 mM NaCl solution suspended with 150 mg/L of clay (D). The pH value of each liquid was adjusted to 5.5, 6.5, 7.5, and 8.5 using either 0.01 N HCl or NaOH. Each sample was spiked with plasmids and E. coli cells, filtrated through 0.2 μm Nuclepore Polycarbonate membrane filter, subjected to DNA extraction, and quantified as described above. Aliquot of 5 × 108 copies of plasmid added into the PowerBead tube was subjected to DNA extraction and used as method control for type A and type B samples. PowerBead tubes containing the same copies of plasmid and 15 mg of clay were processed and used as method control for type C and D samples. Environmental Water Analysis. Storm water runoff samples were collected into sterile plastic bottles from different stormwater discharge channels at the city of Ada, OK after a sustained period of moderate rainfall that produced visible flow in the channels. The absence of the C. parvum 18S rRNA gene fragment was confirmed in these samples by the qPCR procedure described above. Concentrations of Ca2+, K+, and Na+ were analyzed using an inductively coupled plasma optical emission spectrometry (ICP−OES; Optima 3300 DV, PerkinElmer Inc., MA), and standard methods for examination of water and wastewater (APHA, 2005) were followed for determination of pH, TSS, and total heterotrophic bacteria (plate counting techniques) in each stormwater runoff sample. Each sample was then spiked with plasmids to a final concentration of 5 × 106 copies/mL, mixed, and filtrated through the 0.2 μm Nuclepore Polycarbonate membrane filter (Whatman). Plasmid DNA on the filter was quantified using qPCR as described above. An equal number of plasmids spiked directly into a PowerBead tube was subjected to DNA extraction and used as method control. Statistical Analysis. For each test, the percentage recovery of plasmids by filtration (R) was calculated as R = Nsample/ Ncontrol × 100, where Nsample and Ncontrol represent the copy number of plasmids and the corresponding method control, respectively. One-way ANOVA with the posthoc Bonferroni pairwise comparison was performed with SAS software (SAS Institute Inc., 2010) to evaluate the differences of the mean 9326
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Table 1. Mean Percentage Recovery of Spiked Plasmid DNA (in DI Water with pH 7.0) by Filtration Using Membrane Filter of Different Properties mean percentage recovery (±SD, n = 3) of spiked plasmids by membrane filter of different properties type of membrane filter plasmid concentration (copies/mL)
membrane filter pore size, μm
PVDF
5 × 10
0.1 0.2 0.45 0.1 0.2 0.45 0.1 0.2 0.45
13.15 ± 2.67 4.92 ± 1.13 2.56 ± 0.55 17.37 ± 4.84 4.39 ± 1.16 2.77 ± 0.30 14.92 ± 3.79 5.81 ± 1.45 1.88 ± 0.42
4
5 × 105
5 × 106
percentage recovery between (i) the same filter types with different pore sizes, (ii) different types of filters with the same pore size, (iii) suspensions with different TSS contents, (iv) suspensions with different Ca2+ or Na+ concentrations, and (v) suspensions with different pH values. Significance was determined at a P value of less than 0.05.
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PES n/a 5.54 1.96 n/a 5.21 2.07 n/a 4.96 1.90
± 0.81 ± 0.22 ± 1.14 ± 0.09 ± 1.06 ± 0.17
PC n/a 2.81 1.27 n/a 3.38 1.42 n/a 3.07 1.72
± 0.60 ± 0.28 ± 0.88 ± 0.07 ± 1.56 ± 0.71
MCE n/a 14.39 ± 3.92 7.44 ± 4.61 n/a 13.91 ± 4.87 6.91 ± 3.07 n/a 15.55 ± 2.20 7.49 ± 4.29
decreased by the same order; i.e. MCE (6.9%), PVDF (2.8%), PES (2.1%), and PC (1.4%). Impact of TSS. Figure 1 shows the effect of TSS on the percentage recovery of plasmids when passed through a 0.2 μm
RESULTS
The limit of detection for the C. parvum 18S rRNA gene containing plasmid using the qPCR method in this study, as determined by the lowest amount of plasmids that can be quantified, is 90 copies per extraction (equal to about 1.8 copies per PCR reaction). For all tests, IAC always yielded a constant Ct value of around 38, indicating the absence or low concentration of PCR inhibitors in the DNA extracts. The recovery of eDNA refers to DNA trapped on the filter and thus can be quantified by qPCR hereafter. Effects of Membrane Properties. After passing through membrane filters of different types and pore sizes, the mean percentage of recovery (R) of plasmids on filters is shown in Table 1, where R values were generally between 1.3% and 17.4%. For all three concentrations of spiked plasmids, R was affected by both the pore sizes (0.1, 0.2, and 0.45 μm) and the types of membrane filter used. For the same type of filter, R declined substantially with an increase in pore size. Taking the PVDF filter as an example, with a plasmid concentration of 5 × 104 copies/mL, the percentage of recovery was 13.2, 5, and 2.6 for pore sizes of 0.1, 0.2, and 0.45 μm, respectively. After increasing the plasmid concentration to 5 × 105 copies/mL, the percent recovery changed to 17.3, 4.4, and 2.8, respectively. Upon a further increase in the plasmid concentration to 5 × 106 copies/mL, the percent recovery changed to 14.9, 5.8, and 1.9, respectively. As also shown in Table 1, a similar trend of recovery is demonstrated for the other three types of membrane filters. For membranes of the same pore size, regardless of the concentration of the spiked plasmids, the MCE filter always recovered the highest percentage of plasmids, while the lowest recovery was observed using the PC filter. Comparable recovery rates were obtained for both the PVDF and PES filters. For example; when the plasmid concentration was 5 × 106 copies/mL, with a filter pore size of 0.2 μm, the highest recovery was 14% by the MCE filter, 3.4% for the PC filter, and 4.4% and 5.2% for the PVDF and PES filters, respectively. For the 0.45 μm pore-size membranes, the percent recovery
Figure 1. Percentage recovery of plasmid when filtered through 0.2 μm PC filter using water with different total suspended solid. (⧫: inorganic suspended solids, ■: organic suspended solids).
PC filter. The two types of TSS, organic and inorganic suspended solids, differentially impacted the recovery of plasmid by filtration. Compared with a zero TSS control, increases in the concentration of inorganic solids in water sample considerably enhanced the recovery of plasmid DNA. Percentage recovery increased from 3.4% to 6.6% with a solution of 100 mg/L clay. Increasing the clay concentration from 100 mg/L to 1000 mg/L almost tripled the recovery of plasmid DNA. In contrast, the presence of organic particles in suspension only enhanced the recovery of plasmid DNA slightly, while no noticeable increase occurred when changing the organic concentration from 0 to 100 mg/L. Even in the presence of 1000 mg/L organic solids, only 6.4% of the plasmids were recovered by the filtration. Influence of Ionic Strength Components. The influence of Na+ and Ca2+ on the recovery of plasmids when filtration by 0.2 μm PC filter, as shown in Figure 2, was closely related to the presence or absence of clay. For Na+, with the presence of clay (150 mg/L), filtration recovery of eDNA linearly increased when increasing the Na+ concentration in the suspension (R2 = 0.64). Up to 17.3% of plasmid DNA was intercepted on the membrane filter when the NaCl concentration increased to 100 mM. When there was no clay in the solution, an increase of Na+ concentration only slightly increased the recovery of eDNA 9327
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solely reflected the dynamic of added plasmids. As shown in Table 3, chemical analysis indicated that the Ca2+ concentration Table 3. Summary of the Chemical, Physical, and Biological Properties of Stormwater and the Percentage Recovery of Spiked Plasmid DNA
Figure 2. Percentage recovery of plasmid DNA using filtration procedure from water with different cation concentration with or without the presence of clay. Solutions were filtered through 0.2 μm Nuclepore Polycarbonate membrane filter and the plasmid DNA recovered on the filter was extracted and quantified.
5.5 6.5 7.5 8.5
5.1 4.6 3.9 3.6
± ± ± ±
0.2 0.3 0.2 0.3
5 mM NaCl/clay 17.1 15.4 13.7 10.3
± ± ± ±
0.2 0.7 0.4 1.3
DI H2O
DI/clay
± ± ± ±
18.2 ± 0.6 13.2 ± 0.2 10.1 ± 1.5 7.6 ± 1.2
6.3 4.7 3.7 3.8
0.3 0.2 0.2 0.4
K (mM)
Na (mM)
pH
TSS (mg/L)
bacteria (CFU/mL)
% recovery
1 2 3 4 5 6
1.52 1.83 2.11 2.50 2.41 2.65
0.07 0.12 0.07 0.23 0.10 0.11
0.43 0.68 0.51 0.45 0.42 0.23
6.8 7.1 6.5 7.1 6.7 7.3
158 97 262 116 139 107
3700 1120 4100 4600 3900 830
8.3 7.6 12.5 9.7 11.4 6.8
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DISCUSSION The microbiological examination of water is performed routinely by water utilities and governmental agencies to ensure that a safe supply of water is available. Environmental water testing can present challenges that are not frequently encountered in clinical or industrial fields. Because of the small number of targets in a water sample, large volumes must typically be processed in order to increase the target to concentrations that are detectable by routine or molecular biological methods. Filtration is the most commonly used approach in water quality studies and has been successfully proven in concentrating the small density of microorganisms from water samples. Because of its high accuracy and quick processing time, molecular tools, such as qPCR, targeting genetic markers of fecal indicator bacteria are now being more widely used in rapid water testing.5 However, one of the limitations of qPCR is an inability to differentiate genetic targets which originated from viable FIB from extracellular genetic markers, which might produce false-positive data resulting in an overestimation of risk posed to human health. Extracellular DNA occurs in all natural waters and sediments in three forms, including naked free DNA, DNA adsorbed on detrital particles, and protein encapsulated or coated DNA. Despite recent efforts to eliminate the influence of eDNA by incorporating ethidium monoazide bromide or propidium monoazide into procedures,18,24 an accurate interpretation of qPCR signals to health risk is still a challenging task due to the complexity of environmental water. In this paper, we studied variations in the recovery of eDNA in different water samples as a function of filter membrane properties (filter pore size and type) and by mimicking intrinsic
Table 2. Percentage Recovery of Plasmid DNA Spiked in Solution with Different Na+, Clay Concentration and pHa 5 mM NaCl
Ca (mM)
was in the range between 1.5 to 2.7 mM, K+ from 0.07 to 0.23 mM; and Na+ from 0.23 to 0.68 mM. All stormwater samples have a neutral or near neutral pH (value ranged from 6.5 to 7.3). The stormwater samples had a high TSS concentration which ranged from 1.99log10 mg/L to 2.4log10 mg/L, suggesting the inclusion of organic or inorganic suspensions. As determined by a plate counting method, those stormwater samples also contained heterotrophic bacteria with concentrations ranging from 830 to 4,600 CFU/mL. When 5 × 106 copies/mL of plasmids DNA was spiked into stormwater samples and filtrated through 0.2 μm polycarbonate (PC) membrane filter, the recovery ranged from 6.8% to 12.5%. Correlation analysis indicated that the recovery of eDNA was negatively related with water pH value (r = −0.85) and positively associated with the content of TSS (r = 0.75).
through filtration (3.9% to 6.3%). On the contrary, the recovery of eDNA in a CaCl2 solution in the presence of clay exhibited a distinct pattern. With the current experimental setting, an increase in the CaCl2 concentration from 0 to 5 mM linearly increased the recovery of plasmid. Maximum recovery of eDNA (19%) was achieved from a sample with 5 mM Ca2+, and the recovery was significantly reduced when increasing the Ca2+ concentration from 5 to 100 mM. The recovery of eDNA in the CaCl2 only solution showed a similar pattern to that of the NaCl only solution, while increasing the CaCl2 concentration from 0.05 mM to 100 mM only led to a 2% (from 3.9% to 5.9%) rise in the eDNA recovery. Impact of pH. Similar to that of ionic strength components, the impact of pH on the filtration recovery of plasmid DNA by the 0.2 μm PC filter was also strongly associated with the presence of inorganic solids; whereas the ionic components did not seem to interfere with the impact of pH on filtration recovery (Table 2). With the absence of clay in the solution,
pH value
sample no.
Solutions were filtered through 0.2 μm Nuclepore Polycarbonate membrane filter, and the plasmid DNA recovered on the filter was extracted and quantified. a
increasing the DI water pH from 5.5 to pH 8.5 reduced the recovery of plasmid DNA from 6.3% to 3.8%. In the 5 mM NaCl solution with an absence of clay, the recovery of plasmid DNA decreased from 5.1% to 3.6% with a pH increase from 5.5 to 8.5. However, in the presence of 150 mg/L clay, the recovery of plasmids was significantly increased. For example, the recovery of DNA decreased from 17.1% to 10.3% when the NaCl solution pH was raised from 5.5 to 8.5; however, this pH increase without the presence of NaCl decreased the recovery of DNA from 18.2% to 7.6%. Environmental Water Analysis. No C. parvum 18S rRNA gene fragment was detected from stormwater samples before plasmid DNA spiking, indicating that all the changes observed 9328
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which is sufficient to cause misleading management information during microbial water quality assessments. Caution is required when interpreting qPCR data from environmental water with high total suspended solids to avoid overstating the magnitude of pollution. The effect of pH on the sorption of DNA by various research groups demonstrated that it increases at low pH indicating that sorption is a charge dependent process (Table 2). Cai et al. found that the maximum adsorption of salmon sperm DNA on montmorillonite, kaolinite, and goethite occurred at around pH 3.0, possibly due to precipitation on the clay surface caused by the denaturation under acidic conditions.28 Nguyen et al. reported that plasmid DNA molecules are highly negatively charged at pH 6 and 8 due to their phosphate backbone.29 Saeki et al. found that DNA adsorption by andosols was greatly decreased by increasing the pH of suspensions between 3.0 and 9.0.30 It should be pointed out that natural water rarely has extreme pH values. The pH range tested in our study was from 5.5 to 8.5 (Table 2), which is more commonly encountered in environmental water; therefore, the results obtained will be more applicable to environmental studies. In our study, it was found that the influence of ionic components on the recovery of DNA by filtration was closely related to the presence of clay (Figure 2). Nguyen reported that diffusion coefficients for plasmid DNA were enhanced with increasing ionic strength, particularly with respect to Ca2+. The same author suggested that in high-ionic strength solutions, the intramolecular electrostatic repulsion among a negatively charged subsection of the DNA molecules was screened by the ions in solutions.31 The silica surface and the plasmid DNA were negatively charged under the tested pH conditions. With increasing concentrations of monovalent and divalent cations, the shielding of these negative charges substantially decreased the repulsive electrostatic forces thereby facilitating the sorption of DNA to silica. This effect was particularly enhanced in the presence of divalent cations such as Ca2+ which can significantly reduce the charge of clay surfaces by binding to silanol groups present on the surface of clay, thus resulting in reduced electrostatic repulsion between the DNA and the clay surface.29 The binding of the DNA and clay charge neutralization by calcium ions and the increase in the DNA diffusion rate lead to an increase in the sorption rate of the plasmid to the clay surface and thus the enhanced recovery of DNA by filtration. This sorption was shown to be irreversible when rinsing with solutions containing lower ionic strength,29which can explain the V-shape of the recovery curve for plasmid spiked in the Ca2+ solution that was constantly observed in our study. The recovery of DNA was likely controlled by two processes; the cation induced binding of DNA on clay particles before and during filtration and desorption of DNA from colloids during the extraction steps. Although increases in the Ca 2+ concentration caused more DNA binding on colloids, they also increased the binding force between DNA and clay particles which make desorption of DNA from the clay more difficult. The balance between binding and desorption resulted in the observed “higher recovery” but not in the presence of the highest Ca2+ concentration. Studies on the binding of DNA on organic colloids indicated that the process was also associated with ionic strength: with little sorption at low ionic strengths and higher sorption with increasing ionic strength; a phenomenon attributed to electrostatic double layer repulsion between DNA and organic matter.32 Since both DNA and organic matter are negatively
attributes of environmental water (pH, total suspended solids, and salinity) to provide much needed information for the successful implementation of qPCR in monitoring water quality practices. Our data showed that filters fabricated with different materials influenced the recovery of DNA. The MCE filter exhibited the highest potential for DNA recovery, while the PC filter recovered the least (Table 1). The water samples tested in this study lead to the conclusion that the physiochemical nature of the water could also substantially impact the recovery process. Higher DNA was recovered with samples characterized with low pH, high ionic strength, or TSS. eDNA segregated from water was closely associated with the adsorption on the filter membrane (Table 1) and particulate matter (Figure 1), including organic and inorganic suspended solids, heterotrophic bacteria, and large particles. This consistent pattern between recovered DNA and the various factors investigated in this study suggests this is a universal pattern, independent of the length or source of extracellular DNA tested; which should be considered in the application of qPCR strategies. It is important to note, however, that sampling and analytical variations also contribute to observed qPCR deviations.25 Commercially available filters used for water quality monitoring are designed to separate biological or abiotic particulates from water, with the assumption that extracellular DNA passes entirely through the filter membrane; thereby, achieving differentiation between extracellular and intracellular DNA. Our study, however, demonstrated the retention of eDNA by the filtration process even in cases where there were no colloids available for interaction, suggesting that DNA can directly interact with membranes even with those that are designed to have low binding affinities. Therefore, DNA analyses performed by filtration could be accountable for providing misleading data regarding the bacterial composition of the environmental water. Our data suggest that during water quality monitoring practices using qPCR, filters with less retention potential (i.e., PC) are desired to reduce the interference of extracellular DNA and circumvent the possibility of false positive results. Due to the inherent variability of DNA recovery for different filter types, filters with the same properties (type and pore size) need to be retained throughout the entire monitoring to maintain data consistency. We observed a considerable influence of inorganic particles on the recovery of DNA through filtration. Experiments exploring the behavior of DNA have been performed using a variety of substrates such as soils, clay, silica, and natural organic matter. For instance, Lorenz modeled the sorption potential and fate of extracellular DNA in a flow system of sand filled columns under different conditions. It was proposed that the sorption of DNA to sand particles occurred by cation bridging while desorption is enhanced by EDTA and that hydrophobic interactions between DNA and sand play a minor role on DNA sorption.26 Mitra et al. investigated the mechanistic aspect of DNA sorption at a solid−liquid interface. They found that in all cases, the initial rate of sorption is controlled by diffusion following a first order reaction with two kinetic constants which explained the initial attachment of the DNA molecules to the solid surface followed by the rearrangement of DNA.27 Our results agreed with the previous studies and supported the concept that in the presence of colloids more DNA will be absorbed and thus recovered by the filtration process. The percent recovery of spiked plasmid DNA through filtration from stormwater ranged from 7% to 13%, 9329
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results do not necessarily reflect the views of the agency or its policy.
changed, increasing the ionic strength also increases the shielding effects which diminish repulsive conditions, allowing interactions to occur. The observed lower recovery of plasmid DNA from organic matters compared to clay was possibly due to their large particle size and thus the less surface area to volume ratio. The clay particles used in our study have an average size of
