Marine Pollution Bulletin 73 (2013) 47–56

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Marine Pollution Bulletin

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Sample preparation methods for quantitative detection of DNA bymolecular assays and marine biosensors

0025-326X/$ - see front matter Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.marpolbul.2013.06.006

⇑ Corresponding author. Address: NOAA Southwest Fisheries Science Center, LaJolla, CA, USA. Tel.: +1 858 546 7142.

E-mail address: kelly.goodwin@noaa.gov (K.D. Goodwin).1 Current address: University of British Columbia, 2350 Health Sciences Mall,

Vancouver BC V6T-1Z3, Canada.

Annie M. Cox a,1, Kelly D. Goodwin b,⇑a National Oceanic & Atmospheric Administration (NOAA), Northwest Fisheries Science Center, La Jolla, CA 92037, USAb National Oceanic & Atmospheric Administration (NOAA), Atlantic Oceanographic & Meteorological Laboratories (AOML), Miami, FL 33149, USA

a r t i c l e i n f o

Keywords:Sample preparationInhibitionDNA extractionSCODAEnvironmental sample processorESPqPCR

a b s t r a c t

The need for quantitative molecular methods is growing in environmental, food, and medical fields but ishindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts fromEnterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus weretested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, includingSynchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an auto-mated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhi-bition were measured, significantly affecting target quantification. An aggressive lysis method thatutilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commer-cial kit protocols. SCODA purification did not show marked improvement over commercial spin columns.Overall, data suggested a general need to improve sample preparation and to accurately assess andaccount for DNA recovery and inhibition in qPCR applications.

Published by Elsevier Ltd.

1. Introduction

Molecular tools are increasingly being utilized in microbialwater quality assessments. Recent developments that foster thisexpansion include EPA recommendations for qPCR detection of fe-cal indicator bacteria (FIB), quantitative microbial risk assessment(QMRA) and progress with development and validation of quanti-tative microbial source tracking (MST) (Wade et al., 2010; US EPA,2010a, 2010c; Roslev and Bukh, 2011; US EPA, 2012; Boehm et al.,in press). Water quality monitoring and public notification ofhealth threats could be further improved by rapid and automatedbiological sensing systems. Coupled with environmental informa-tion, biosensors could be used to enhance assessments and fore-casts for a variety of applications including beach warnings,shellfish closures, harmful algae warnings, and commercial fishingpredictions based on larval abundances (Kröger et al., 2002; Krögerand Law, 2005; Goffredi et al., 2006; Greenfield et al., 2006; Paulet al., 2007; Scholin et al., 2009). The economic benefits of suchocean observing systems have been estimated at $274.7 M/yr forrecreational waters (beaches) and �$150 M/yr for recreationaland commercial fisheries (Kite-Powell et al., 2008). The Environ-mental Sample Processor (ESP) developed by the Monterey Bay

Aquarium Research Institute (MBARI) (Preston et al., 2009, 2011)is a commercially available ‘‘ecogenomic sensor’’ (Scholin, 2010).The ESP can filter particulates from water samples and lyse themfor in situ analyses by qPCR.

Quantitative PCR (qPCR) is attractive for water quality monitor-ing (US EPA, 2010a) because current culture-based methods forpathogens and fecal indicator bacteria (US EPA, 2004; DePaolaand Kaysner, 2004; Thompson et al., 2005) are too slow (Nobleand Weisberg, 2005). Methods for enterococci and many patho-gens have been developed (Haugland et al., 2005; Siefring et al.,2008; Nordstrom et al., 2007), with water quality criteria formolecular detection of enterococci recently made available (USEPA, 2012). However, low and variable extraction efficiencies andPCR inhibition (Santo Domingo et al., 2007; Goodwin and Litaker,2008; Stewart et al., 2008; Goodwin et al., 2009; Stoeckel et al.,2009) may hamper qPCR for water quality monitoring (Schrieweret al., 2011; Green and Field, 2012).

PCR reactions can be inhibited by humic and fulvic acids, poly-saccharides, metal ions (Kreader, 1996; Wilson, 1997; Watson andBlackwell, 2000; Kermekchiev et al., 2009; Opel et al., 2010; Schrie-wer et al., 2011; Haugland et al., 2012), and large quantities of non-target DNA (Ludwig and Schleifer, 2000). Recognition of inhibitioncan be achieved through dilution experiments (Schriewer et al.,2011; Cao et al., 2012), amplification of a control such as salmontestes DNA (Wade et al., 2010) or synthetic DNA (Nordstromet al., 2007; Noble et al., 2010; Pontiroli et al., 2011), or with dilu-tions of a spike of the target DNA (Haugland et al., 2005; Yamahara

48 A.M. Cox, K.D. Goodwin / Marine Pollution Bulletin 73 (2013) 47–56

et al., 2009; Cao et al., 2012). Once inhibition is identified, attemptsto overcome inhibition typically involve chemically blocking inhib-itor activity (Kreader, 1996), diluting the extract (Haugland et al.,2005; Siefring et al., 2008; Schriewer et al., 2011) or further purifi-cation (He and Jiang, 2005). However, additional processing stepscan result in DNA loss (Roose-Amsaleg et al., 2001; Schrieweret al., 2011) and dilution lowers the number of cells delivered tothe qPCR reaction, thus lowering the sensitivity of the method. Fil-tering larger amounts of water has been used in an attempt to in-crease the amount of DNA target; however this tends to becounterproductive because it also concentrates more PCR inhibi-tors (Loge et al., 2002; Thompson et al., 2005) and increases theamount of nontarget DNA.

Some studies have undertaken the issue of DNA extraction inseawater (Fuhrman et al., 1988; Somerville et al., 1989; Boccuzziet al., 1998; Boström et al., 2004; Simmon et al., 2004). Extractionefficiencies in seawater typically were found to be less than 50%(Fuhrman et al., 1988; Rivera et al., 2003), similar to that foundwith soil (Mumy and Findlay, 2004; Smalla et al., 1993). One studyreported high recovery of target from seawater (92–96%) (Boströmet al., 2004); however, it appears that this method has yet to betested thoroughly on PCR-inhibited samples. Inhibition poses abarrier to implementation of qPCR with environmental samples(Goodwin and Litaker, 2008; Stewart et al., 2008, in press; Stoeckelet al., 2009; Noble et al., 2010; Schriewer et al., 2011). Withoutinhibition, measured qPCR values typically are higher than culturevalues (Haugland et al., 2005; Byappanahalli et al., 2010; Whitmanet al., 2010; Converse et al., 2012). In general, an inability to fullyreconcile culture counts to qPCR (Ludwig and Schleifer, 2000) hin-ders application of qPCR technology to water quality monitoring.

This study explored the effect of DNA extraction protocols onqPCR with regard to the number of target copies recovered andthe level of inhibition. The goal was to explore novel sample prep-aration methods and elements of standard protocols to betterunderstand the effect of DNA sample preparation on qPCR, in gen-eral, and improvement options for the ESP, in particular. In addi-tion to a variety of kits and combinations of lysis and purificationmethods, a novel electrophoretic DNA purification method, Syn-chronous Coefficient of Drag Alteration (SCODA), was tested. SCO-DA is a multidimensional electrophoretic method that selectivelyconcentrates DNA while rejecting known contaminants (Broemel-ing et al., 2008). In SCODA, rotating electric fields concentrateDNA in the center well of an agarose gel while contaminants, suchas negatively charged humic substances, move outward into wastebuffer (Broemeling et al., 2008; Pel et al., 2009). In addition, testingincluded sample purification on a microfluidics block, a componentof the ESP available for bench-top use, and sample lysis on a fullESP.

2. Materials and methods

2.1. Experiments using cultures

Experiments with cultured cells used Enterococcus faecium orVibrio parahaemolyticus TX2103. E. faecium (ATCC 19434) wasgrown in brain–heart infusion broth (EMD Chemicals; Gibbstown,NJ) and V. parahaemolyticus (RIMD2210633) was grown in trypticsoy broth (Becton, Dickinson and Co.; Sparks, MD). Cells weregrown overnight at 37 �C with shaking (175 rpm) to an approxi-mate concentration of 109 cells/mL. Except for ESP experimentswhich used whole culture, cells were washed three or four timesin relatively large volumes of phosphate buffered saline (PBS, typ-ically 1:50 mL). Cell cultures were serially diluted in PBS to makeappropriate concentrations. Cell pellets were created by centrifug-ing 1 mL of cell solution (10,000g, 5 min). Bacteria cultures were

enumerated by standard serial dilution onto the growth mediaand/or by microscopy. Samples to be stained and enumerated bymicroscopy (Hobbie et al., 1977) were preserved in 15% glycerolat �80 �C until used. DAPI stain (40,6-diamidino-2-phenylindoledihydrochloride; Sigma, St. Louis, MO) generally was used,although acridine orange (Hardy Diagnostics, Santa Maria, CA)with detergent (Tween 80; Sigma, St. Louis, MO) to remove clump-ing also was tried. Counts enumerated by DAPI microscopy are pro-vided here unless otherwise noted.

2.2. Experiments using seawater that inhibited PCR reactions

Protocols were tested on seawater samples in which PCR reac-tions were inhibited (hereafter termed ‘‘PCR-inhibited seawater’’).PCR-inhibited seawater primarily consisted of samples concen-trated (100 L down to 400 mL) by the Portable Multi-use Auto-mated Concentration System (PMACS 1000™), which utilizeddead-end hollow-fiber ultrafiltration (Leskinen and Lim, 2008).PMACS-concentrated seawater was collected from the Ben T. Davisbeach (Tampa, FL) on 26 October, 2010 and the retentate stored at�80 �C until used. For testing various full sample preparationmethods (lysis plus purification), replicate cell pellets were createdby centrifuging (10,000g, 5 min) 1 mL of the PMACS 1000™ con-centrated seawater. For various tests involving separation of lysisand purification steps, 250 lL of PMACS 1000™ concentrate wasfiltered onto 25 mm, 0.22-lm membrane filters (Durapore#GVWP02500, Millipore; Billerica, MA). PCR-inhibited seawatersamples also were collected from Del Mar North Beach (Del Mar,CA) on 27 September, 2011 and processed by standard membranefiltration. This seawater was first filtered through a 20-lm meshprior to filtration of 10 mL (each) through 25 mm, 0.22-lm Dura-pore membrane filters. This seawater sample was used to comparethe performance of a variety of spin column kits (GeneRite DNA-EZ,MO BIO PowerSoil�, PowerWater�, and PowerWater� Sterivex;Table 1).

2.3. qPCR analyses

All qPCR standards and samples were run in duplicate on aMX3005P Real-Time PCR System (Stratagene, La Jolla, CA). Geno-mic DNA for standard curves was quantified using a spectropho-tometer (Nanodrop ND-1000; Wilmington; DE). For Enterococcusspp., PCR primers and probe to amplify a fragment of the 23S (largesubunit) rRNA gene consisted of forward primer ECST748F(50-AGAAATTCCAAACGAACTTG), reverse primer ENC854R (50-CAGTGCTCTACCTCCATCATT), and probe GPL813TQ (50-6FAM-TGGTTCTCTCCGAAATAGCTTTAGGGCTA-Black Hole Quencher 1)(IDT; Coralville, IA) (Ludwig and Schleifer, 2000; Haugland et al.,2005). In this study, reaction mixtures consisted of 15 lL Accu-Prime SuperMix I (Invitrogen; Carlsbad, CA), 1 lM forward and re-verse primers, 0.25 lM probe, 1 mM additional MgCl2 (Sigma; St.Louis, MO), 6 lL template DNA, and DNAse-free water for a finalreaction volume of 30 lL. Thermal cycling conditions consisted of94 �C for 2 min followed by 45 cycles of 94 �C for 15 s and 60 �Cfor 1 min. Conversion calculations to 23S rRNA copy number werebased on an E. faecium genome size of 2.6 Mb and 6 rRNA operonsper cell (Oana et al., 2002). A standard curve was run on everyplate, and 10-fold concentrations were run in triplicate rangingfrom 1.26 � 100 to 1.26 � 105 copies per reaction mixture.

For V. parahaemolyticus, PCR primers and probe to amplify a frag-ment of the thermolabile hemolysin (tlh) gene consisted of tlhforward (50-ACTCAACACAAGAAGAGATCGACAA), tlh reverse(50-GATGAGCGGTTGATGTCCAA), and tlh probe (50-Tx Red-CGCTCGCGTTCACGAAACCGT-Black Hole Quencher 2) (IDT; Coralville,IA) (Nordstrom et al., 2007). Conversion calculations to gene copynumber were based on a genome size of 5.17 Mb and one copy of

Table 1Summary of basic methods used in this study.

Abbreviation Method Lysisa Purification Reference or Company

BEA Bead beating (crude lysis) B None unless noted Haugland et al., 2005BOR Boreal soil lysis B,H,E,C Varied Boreal Genomics; Los Altos, CASCO SCODA purification Variedb Electrophoretic Boreal Genomics; Los Altos, CAAUR Aurora purification B,H,E,Cc Electrophoretic Boreal Genomics; Los Altos, CABOS DNA precipitation w/tRNA C,E Alcohol precipitation Boström et al., 2004 (centrifugation option)CLA PureLyse� Kit Bd Zirconium-silica beads column Claremont BioSolutions; Upland, CAESP ESP bench lysis/SPE (Solid Phase Extraction) H,C Glass beads column Preston et al., 2011GEN GeneRite DNA-EZ ST1 kit B,C Silica column GeneRite; North Brunswick, NJMOB PowerSoil� DNA Isolation kit B,C Silica column MO BIO Laboratories; Carlsbad, CAMOB/PW PowerWater� DNA Isolation kit B,C Silica column MO BIO Laboratories; Carlsbad, CAMOB/PWS PowerWater� Sterivex kit B,H,C Silica column MO BIO Laboratories; Carlsbad, CAQIA DNeasy Blood and Tissue C,Ee Silica column Qiagen; Valencia, CA

a B = bead beating, H = heat, E = enzymatic, C = chemical (detergents, guanidium isothiocyanate).b Typically Boreal soil or Boreal syringe lysis. The Boreal syringe lysis protocol used enzymes and syringe shearing; lysate was sometimes desalted with an Amicon column.c Used Boreal soil protocol.d Micro-Bead-Beater™ included with kit.e Pre-treatment for Gram-positive bacteria protocol.

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the tlh gene for strain RIMD2210633 (Makino et al., 2003). Reactionmixtures consisted of 12.5 lL AccuPrime SuperMix I (Invitrogen;Carlsbad, CA), 75 nM forward and reverse primers, 150 nM probe,1 mM additional MgCl2 (Sigma; St. Louis, MO), 2 lL templateDNA, and DNAse-free water for a final reaction volume of 25 lL.Thermal cycling conditions consisted of 94 �C for 2 min followedby 45 cycles of 95 �C for 15 s and 59 �C for 1 min. A standard curvewas run on every plate, and 10-fold concentrations were run in trip-licate ranging from 1.0 � 100 to 1.0 � 106 copies per reactionmixture.

Actual true final concentrations of enterococci in the concen-trated seawater samples (see Section 2.2) were unknown. There-fore, all results were compared based on the number of copiesrecovered in the DNA extract, which was calculated as the copieslL�1 value according to qPCR multiplied by the extract volume(eluent or crude lysate). Where noted for evaluation of the Aurorasystem, the concentration also was compared. The concentration inthe original volume of sample (1 ml) was equivalent to the copiesrecovered except for extracts produced by the GeneRite kit accord-ing to the manufacturer’s instructions (catalog# K200-01C; Gene-Rite; North Brunswick, NJ). To obtain a value for concentration inthat case, the copies in the original sample were multiplied by acorrection factor of two to account for the fact that only a portionof the lysate was carried through to purification (200 ll clarifiedlysate/400 ll lysate) (GeneRite, personal communication; Ebentieret al., in press; Schriewer et al., in press).

2.4. Calculation of inhibition factors

Sample inhibition was estimated by comparing values fromqPCR reactions containing undiluted and diluted sample DNA. Cy-cle threshold (Ct) values for undiluted DNA were compared toreactions containing diluted DNA (1:10 as well as a 1:25 or1:100 dilution) to assess if DNA dilution caused the Ct value to de-crease (i.e., produce more signal) greater than expected from a lin-ear dilution. Sometimes larger dilutions caused the bacterialconcentration to be diluted below the lowest standard; therefore,comparison to only the 1:10 dilution was made to allow for consis-tent analysis across experiments. An inhibition factor (IF) was cal-culated as Copiesud/Copies1:10, where Copiesud was the copynumber recovered from the undiluted DNA, and Copies1:10 wasthe dilution-corrected final copy number recovered from the1:10 diluted DNA. A sample with IF P 0.81 was considered notinhibited, IF < 0.81 as inhibited, and IF = 0 as fully inhibited. A per-fect dilution would give an IF = 1; however, a value of 0.81 wasused to account for inherent variability and was calculated given

a precision of 0.24 based on the Enterococcus spp. qPCR assay asa guide (log10 standard deviation; Siefring et al., 2008; US EPA,2010b). Inhibition factors were not reported for samples in whichdilution caused the target concentration to fall below the lowestconcentration of the standard curve or for samples with IF values>1.32 (i.e., >0.24 precision).

2.5. Lysis and purification methods overview

Thirteen lysis and purification methods were tested hereaccording to the manufacturer’s instructions or the referenced pa-pers (Table 1), unless otherwise specified. In general, methodswere directly compared to each other based on the metric of copiesrecovered, calculated as the copies per lL�1 of qPCR reaction mul-tiplied by the eluent volume. Protocols that were not readily avail-able elsewhere (ESP and Boreal methods) are briefly describedbelow.

2.5.1. ESP lysis and purificationThe core ESP automatically filters a water sample on a robotic

puck system and lyses the sample on the filter (Scholin et al.,2009). The microfluidics block (MFB) is an ESP component that dis-tributes lysate and reagents to other analytical modules such as thesolid phase extraction (SPE) column for DNA purification and to theqPCR module for amplification (Preston et al., 2009, 2011; Scholinet al., 2009). The SPE column used for DNA purification consists ofreusable glass beads. The MFB equipped with SPE can be obtainedas a stand-alone unit for bench-top use with or without the ESPand qPCR modules. Experiments with the standalone MFB unitused here were performed at the Southern California Coastal WaterResearch Project (SCCWRP) in Costa Mesa, CA.

The ‘‘ESP bench protocol’’ was developed at MBARI to mimic ly-sis on the full ESP (Preston et al., 2011). In the experiments here,lysis buffer (3 M guanidine thiocyanate, 50 mM Tris, 15 mM EDTA,2% N-lauroylsarcosine, 0.2% sodium dodecyl sulfate; pH 8.9; all re-agents from Sigma; St. Louis, MO) was added to a sample pellet orfilter and incubated at 85 �C for 10 min, with vortexing at 5 min.The standard ESP bench protocol called for incubation in 1.4 mLof lysis buffer; whereas, the protocol used on the MFB unit atSCCWRP was modified to maximize DNA recovery such that thesample was incubated with 300 lL of lysis buffer. After incubation,the lysate was filtered through a 0.22-lm, 13 mm syringe filter(Millex-GV, Millipore) and 250 lL of lysate was mixed with225 lL of Solid Phase Extraction (SPE) diluent (sodium acetate(5 M, pH 5.2): 89% ethanol, 1:1 by volume). For experiments usingthe stand alone MFB, the lysate/SPE diluent mixture was fed into

50 A.M. Cox, K.D. Goodwin / Marine Pollution Bulletin 73 (2013) 47–56

the MFB for SPE purification. If the MFB was not used, the lysate/SPE diluent mixture was typically purified using a Qiagen columnfrom a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA),although a number of purification variations were explored (Tables1–3).

2.5.2. Boreal syringe and soil protocols with SCODA purificationDNA lysis for use with SCODA system purification typically uti-

lized a syringe protocol provided by Boreal Genomics (protocol,14D05.1; So et al., 2010). As used here, the method consisted ofadding 100 lL of BLB buffer (50 mM tris–HCl, 50 mM Na2EDTA,0.5% sodium dodecyl sulfate (SDS); pH 7.5; all from Sigma, St.Louis, MO), 1 lL of RNase A (4 mg/mL, Sigma, St. Louis, MO), 1 lLof lysozyme (50 mg/mL, Sigma, St. Louis, MO), and 1 lL of protein-ase K (20 mg/mL; Invitrogen, Carlsbad, CA) to each cell pellet. Sam-ples were incubated (20 min, 37 �C) then solubilized and shearedby passing the lysate 10 times through a 21 gauge � 2’’ needle(Becton, Dickinson and Co., Sparks, MD) attached to a 1 mL syringe(Becton, Dickinson and Co., Sparks, MD).

Lysates from the syringe protocol were tested with and withoutdesalting. Desalting was used to decrease sample conductivityprior to SCODA purification because the technique requires sampleconductivity less than 100 lS/cm (Boreal Genomics, personal com-munication). For desalting, samples were centrifuged through Ami-con Ultra 0.5 mL centrifugal filter devices (30 kDA cut-off,#UFC503024, Millipore, Billerica, MA) followed with two 500 lLrinses of PCR-grade water. The �30 lL of sample remaining inthe Amicon was removed and pooled with a 470 lL PCR-gradewater rinse of the Amicon filter. The sample was brought to 1 mLwith PCR-grade water.

Table 2Description of lysis methods adapted to hold the final purification step (GeneRite Kitsilica column) constant. Abbreviations correspond to Fig. 3a. See Table 1 for additionalinformation.

Abbreviation Lysis protocol Binding buffera (lL added)

BEA Bead beating GeneRite (540)BOR Boreal Genomics soil GeneRite (2700)b

ESP ESP bench SPE Diluent (225)GEN GeneRite GeneRite (600)MOB Mo BIO PowerSoil� GeneRite (2250)c

QIA Qiagen DNeasy Blood andTissue

Buffer AL (200), ethanol(200)

a GeneRite binding buffer added to lysate 3:1.b Followed by Amicon desalting.c Lysis protocol followed up to the point where Solution C4 would have been

added.

Table 3Description of purification methods adapted to hold the lysis method constant(GeneRite Kit lysis protocol). Abbreviations correspond to Fig. 3b. See Table 1 foradditional information.

Abbreviation Binding buffer (lL)a Final purification method

BOS N/A Alcohol precipitationGEN GeneRite (600) GeneRite DNA-EZ silica columnGEN/tb GeneRite (600) GeneRite DNA-EZ silica columnMOB Solution C4 (320) MO BIO PowerSoil� silica columnQIA Buffer AL (200) and

ethanol (200)Qiagen DNeasy Blood and Tissuesilica column

QIA/tb Buffer AL (200) andethanol (200)

Qiagen DNeasy Blood and Tissuesilica column

SCO N/A SCODA electrophoreticSPE SPE diluent (180) ESP MFB Solid Phase Extraction

a Binding buffer was added to lysate according to the proportion described foreach method that uses a column.

b 50 lg yeast tRNA added before the sample was applied to the column.

The sample lysis for soil protocol (‘‘Low Molecular Weight DNADirect from Soil Protocol’’) was provided by Boreal Genomics(termed here as ‘‘Boreal soil lysis’’). The method was a modificationof Zhou et al. (1996) and consisted of adding 1.08 mL of preheated(37 �C) DNA extraction buffer (1 M Tris–HCl pH 8.0, 0.5 M diso-dium EDTA pH 8.0, 1 M sodium phosphate pH 8.0, 5 M NaCl, 10%cetyl trimethylammonium bromide [CTAB]; all from Sigma, St.Louis, MO) to each pellet or filter sample. Tubes were taped to ashaking incubator (225 rpm) and incubated at 37 �C for 10 min.Next, 12.5 lL proteinase K (20 mg/mL; Invitrogen, Carlsbad, CA)and 120 lL SDS (20%; Sigma, St. Louis, MO) were added and vor-texed to mix. Samples were bead beat (5 cycles at 6 m/s, 1 min/cy-cle; FastPrep FP120, Qbiogene, Carlsbad, CA), incubated (65 �C,10 min), and centrifuged (14,000g, 3 min). The supernatant was re-moved, the sample was centrifuged again (14,000g, 5 min), and theremaining supernatant was removed. Samples used for SCODApurification were desalted by adding the supernatant to a 50 mLfalcon tube, filling the tube with 30 mL PCR-grade water, and mix-ing back and forth 10 times between another falcon tube contain-ing 30 mL of PCR-grade water. The sample was added to an AmiconUltra-15 centrifugal filter device (10 kDA or 30 kDA cut-off,#UFC901008 or #UFC903008; Millipore; Billerica, MA) 15 mL at atime and centrifuged (4000g, 15 min), discarding the flow through.After the entire sample (60 mL) was passed through the device, anadditional 15 mL of PCR-grade water was added and centrifuged(4000g, 15 min). The �200 lL of sample remaining in the Amiconwas removed and pooled with an 800 lL PCR-grade water rinseof the Amicon device and brought up to 5 ml with PCR-gradewater. Experiments using desalted lysate are specified as ‘‘AMI’’in figures or tables.

For DNA purification with the SCODA system, lysate was placedin the SCODA injection chamber. The SCODA system utilized across-shaped plastic gel boat consisting of a center area for a smallagarose gel (1.5 � 1.5 cm) surrounded by four chambers for bufferand an injection chamber located to the right of the gel (Broemel-ing et al., 2008). The injection chamber could be created for a 1 mLor 5 mL volume depending on placement of the agarose gel dam(1 ml was used for the Boreal syringe protocol, 5 ml was used forthe Boreal soil protocol). Agarose (1%, SeaKem LE agarose, Lonza,Rockland, ME) was poured into the gel boat and allowed to solidifyaround a metal tube centered in the gel. The tube was removedfrom the solidified agarose, creating a small diffusive extractionwell in the gel center. Buffer chambers and the extraction wellwere filled with buffer (0.25� TBE) and the extraction well wascovered with plastic sealing film. SCODA set up used DevelopmentSystem 1.4 as per the manufacturer’s instructions with the follow-ing optimized parameters: injection voltage: 90 V (30 min), con-centration/wash: SCODA field strength 45 V/cm, 4 s cycle period(1.5 h), final concentration: SCODA field strength 45 V/cm, 4 s cycleperiod (2 h). At the end of the SCODA concentration and purifica-tion run, the purified DNA was removed from the center welland could be used directly for PCR.

2.6. Separation of lysis and purification steps

Typical DNA extraction protocols consist of lysis and purifica-tion steps in composite; therefore, protocols were split to assessthe impact of lysis and purification on overall recovery of DNA.To compare lysis procedures, the purification step was held con-stant (and vice-versa to compare purification steps). Samples con-sisted of PMACS 1000™ concentrate (250 lL) filtered onto 25 mm,0.22-lm Durapore membrane filters (see Section 2.2).

Various lysis methods (Table 2) were compared using the Gene-Rite column, the Qiagen column, or SCODA as the common purifi-cation method. For GeneRite purification experiments for thoseprotocols without column purification (e.g., bead beating (BEA),

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Boreal soil lysis (BOR)), the binding buffer provided with the Gene-Rite kit was added to the lysate in the proportion described as perthe manufacturer’s instruction (3:1 binding buffer to lysate ratio tomatch the 600:200 lL ratio of the instructions; see Table 2). For theremainder of the procedure, the GeneRite column and wash bufferwas used following GeneRite kit instructions.

Six purification methods (Table 3) were compared using theGeneRite kit lysis instructions as the common lysis method. Gene-Rite was chosen because kit performance with environmental sam-ples was such that it was chosen for a recent inter-laboratorycomparison of quantitative microbial source tracking methods(Boehm et al., in press). The protocol was followed to the point inwhich the binding buffer would have been added to the spin col-umn. At that point, the additive specified in the purification proto-col being tested was added to the GeneRite lysate (200 lL) in theproportion described for that protocol (Table 3) and the protocolwas followed from there without further modification. For certainGeneRite and Qiagen column purification trials, 50 lg yeast tRNA(Ambion, Grand Island, NY) was added to the lysate prior to bindingDNA to the column to determine if this would increase DNA yield.Tests of SCODA purification used desalted lysate (see Section 2.6.2).

2.7. ESP experiments

Experiments with a full ESP were performed at the NOAANorthwest Fisheries Science Center (Seattle, WA). Cell cultures ofE. faecium and V. parahaemolyticus (see Section 2.1) were dilutedin PBS and spiked into seawater inhibited by adding sediment(both collected from Golden Gardens Park, Seattle, WA on 31 Octo-ber, 2011 and 3 November, 2011) to make a final cultured cell con-centration on the order of 103 cells/mL. Samples were processed(collected, filtered, and lysed) by the ESP short lysate protocol(bacsh1) according to the manual (version 2.1) and instructionsby Spyglass, Inc. (personal communication). Samples (10 mL) alsowere processed simultaneously by membrane filtration (25 mm,0.22 lm Durapore) for comparison to samples processed by theESP. The membrane-filtered samples were stored on ice and lysedusing the GeneRite kit while the samples on the ESP were lysed. Forboth ESP-processed and membrane-filtered samples, lysates(1.4 mL) from three filters were composited to minimize variabilityand composites were analyzed in triplicate by qPCR (therefore eachexperiment represents a total of 9 filters). Composited lysates fromthe ESP and from membrane-filtered samples (200 lL) were puri-fied with the GeneRite kit as per the manufacturer’s instructionsand run by qPCR in Seattle (see Section 2.3; both NOAA facilitieshouse Stratagene MX3005P instruments).

2.8. Statistical analysis

Statistical differences between treatment means were deter-mined using one-way analysis of variance (ANOVA) with Tukey’sHSD post hoc test. Data were analyzed using R Statistics (version2.13.2, R Development Core Team, 2011).

3. Results

Standard curve metrics indicated that both qPCR assays per-formed adequately (Stratagene, 2004), with results as follows:Enterococcus: 92–104% efficiency, 0.970–1.00 r2, and 39.34–43.56y-intercept. Vibrio: 94–106% efficiency, 0.990–0.999 r2, and y-inter-cept 44.18–45.58. The lower limit of quantification (LLOQ) for theEnterococcus assay was considered to be 12.6 copies/reaction be-cause amplification occurred in 100% of trials (37.72–39.77 Ct),whereas the 1.26 copies/reaction standard amplified less than50% of the time. The Vibrio assay appeared to be less sensitive;

amplification of the 100 copies/reaction occurred in 100% of thetrials, the 10 copies/reaction standard in >50%, and none of the1 copy/reaction standards amplified.

In all experiments, methods (Table 1) were compared directlybased on the number of target copies recovered (copies per lL�1

multiplied by eluent volume). Recovery of target relative to enu-meration by culture or microscopy was not possible for the frozenconcentrated seawater samples. In addition, defaulting to this met-ric for comparison avoided the difficulties of comparing qPCR toother types of enumeration (Ludwig and Schleifer, 2000). Forexample, DAPI counts for experiments that used cultures (as inFigs. 1 and 5) typically were an order of magnitude higher thancorresponding plate counts, with similar variability (percent coef-ficient of variation, %CV: 13% DAPI, 20% plate counts). Enumerationby qPCR was higher than microscopy. Washing cell pellets threetimes decreased the relative overage but did not eliminate it fully.

3.1. Target recovery from cultures and PCR-inhibited seawater

Recovery and variability differed among methods, sometimesby more than an order of magnitude (Figs. 1–3, Table 4). The BOSmethod described by Boström et al. (2004) provided the highestrecovery for E. faecium culture spiked into PBS, and recovery wassignificantly higher than the other tested methods except for beadbeating (BEA), bead beating with SCODA purification (BEA/SCO),and Claremont BioSolutions (CLA) (p < 0.05; Fig. 1A–C, note: beadbeating and bead beating with SCODA purification were testedonly with the 105 concentration of E. faecium). In contrast, the Bos-tröm method for DNA extraction (BOS) provided no target copieswhen used with undiluted extracts of concentrated seawater(PMACS 1000™ system). Instead, the GeneRite kit (GEN) providedthe highest copies recovered compared to other tested methodsand was significantly different to all except bead beating(p < 0.05; Fig. 2a and Table 4).

Due to encouraging recovery and inhibitor removal with theconcentrated seawater (Fig. 2a), the GeneRite kit was comparedto three MoBio kits using filtered seawater (Fig. 2b). In this case,recovery with the GeneRite kit (GEN) (1.73 � 103 copies recovered)was three times higher than the MO BIO PowerSoil� (MOB) andPowerWater� kits (MOB/PW) (548 and 649 copies recovered,respectively) and thirty times higher than the PowerWater� Steri-vex kit (MOB/PWS) (58 copies recovered; Fig. 2b). These differ-ences between GeneRite and the three MO BIO kits weresignificant, (p < 0.05). The %CV values were similar (29–37%) forthese methods, except for higher variability observed for the Pow-erWater� Sterivex kit (106%).

3.2. The effect of inhibition on target recovery

Inhibition factors were analyzed to investigate whether the ob-served differences in method performance between samples of cul-ture (Fig. 1) and seawater (Fig. 2) resulted from inhibition.Consistent with this hypothesis, GeneRite and MO BIO PowerSoil�

kits showed low inhibition (Fig. 2a, Tables 4 and 5) and relativelybetter recovery with environmental samples than with samplesof culture. In contrast, no copies were recovered from the undi-luted extracts produced by the Boström method (BOS; Fig. 2a),although this method showed the highest recovery with culturespiked into PBS (Fig. 1).

Sample inhibition affected quantification, with dilution increas-ing the estimated copies recovered for inhibited samples sometimesby more than an order of magnitude (Table 4). This effect could onlybe observed if samples contained sufficient target to withstand dilu-tion. For example, filtered seawater samples used to compare MoBiokits also showed some inhibition (Fig. 2b); however, dilution gener-ally reduced Ct values below that of the lowest standard.

Method

Cop

ies

reco

vere

d

A104

Method

Cop

ies

reco

vere

d

B105

Method

Cop

ies

reco

vere

d

C106

Fig. 1. Copies recovered from undiluted DNA extracted from E. faecium culture at concentrations of (A) 104, (B) 105, and (C) 106 copies pellet�1; n = 3 for each method exceptn = 5 for MOB and ESP of A). Method abbreviations and descriptions provided in the text and Table 1.

Method

A

Cop

ies

reco

vere

d

0.32

0.00 0.00

0.100.80

0.29

0.90

Method

Cop

ies

reco

vere

d

0.73

B

Fig. 2. Box and whisker plots comparing the number of enterococci copies recovered from undiluted DNA extracted from concentrated seawater using various samplepreparation methods. DNA was extracted from (A) seawater concentrated by PMACS 1000™ and pelleted (n = 5) or (B) seawater concentrated by membrane filtration (n = 6,except for GEN, n = 9). The average inhibition factor is given above each box except for cases in which dilution lowered the Ct value below that of the lowest standard. SeeTable 1 for abbreviations (Fig. 2a: SCO = Boreal syringe lysis with SCODA purification).

Method

Cop

ies

reco

vere

d

(B) Purification constant0.54

0.51

0.13

0.79

0.46

0.42

Method

Cop

ies

reco

vere

d

0.00

0.83

0.00

(A) Lysis constant 0.50

Fig. 3. Box and whisker plots comparing the number of enterococci copies recovered from undiluted DNA extracted from filters (n = 3 each) of concentrated seawater usingsample preparation methods in which (A) purification was varied with GeneRite lysis constant (see Table 3), and (B) lysis was varied with GeneRite purification held constant(see Table 2). The average inhibition factor for an individual experiment is given above each box except for cases in which dilution lowered the Ct value below that of thelowest standard.

Table 4qPCR results for different dilutions of DNA extracted from PCR-inhibited seawater by various methods.

Method Target copies recovered (%CV, n)a

1:1 1:10 1:100

GeneRite DNA-EZ ST1 Kit 2.25 � 105 (15%, 5) 2.64 � 105 (24%, 5) 2.97 � 105 (60%, 5)Bead beating 1.81 � 105 (26%, 5) 6.11 � 105 (19%, 5) 4.31 � 105 (68%, 5)MO BIO PowerSoil� DNA Isolation Kit 7.26 � 104 (4%, 5) 8.01 � 104 (33%, 5) 5.18 � 104 (25%, 5)Bead beating lysis/SCODA purification 3.43 � 104 (110%, 4) 2.62 � 105 (39%, 5) 2.52 � 105 (50%, 5)Boreal soil lysis/SCODA purification 2.67 � 103 (18%, 3) 9.82 � 104 (107%, 4) 1.33 � 105 (173%, 4)Boström lysis with tRNA co-precipitation 0 0 3.05 � 105 (76%, 3)Bench ESP lysis/MFB-SPE purification 0 2.25 � 103 (8%, 5) 6.60 � 103 (23%, 5)

a Based on n amplified extracts.

52 A.M. Cox, K.D. Goodwin / Marine Pollution Bulletin 73 (2013) 47–56

3.3. The effect of lysis and purification methods on target recovery

Given the differences in recovery measured between DNAextraction protocols, method components were separated to inves-

tigate whether lysis or purification drove overall recovery and var-iability and to consider possible opportunities to improve recovery.Purification steps were held constant to compare lysis protocolsand vice-versa to compare purification protocols (Figs. 3 and 4).

Table 5Average inhibition factors (IF) for selected methods in this study using concentratedseawater samples.

Method IF ± STD (n)

GeneRite DNA-EZ ST1 Kit 0.85 ± 0.21 (9)MO BIO PowerSoil� DNA Isolation Kit 0.80 ± 0.10 (4)MO BIO PowerWater� DNA Isolation Kit 0.73 ± 0.26 (3)Qiagen DNeasy Blood and Tissue Kit 0.43 ± 0.07 (3)Boström lysis with tRNA co-precipitation 0.0 ± 0.0 (3)b

Bead beating (crude lysis) 0.32 ± 0.17 (8)Bead beating/GeneRite column purification 0.51 ± 0.25 (3)Bead beating/SCODA purificationa 0.38 ± 0.38 (9)Boreal syringe lysis/SCODA purification 0.29 ± 0.46 (4)GeneRite lysis/SCODA purification 0.83 ± 0.18 (2)Boreal soil lysis/SCODA purification 0.53 ± 0.15 (4)Boreal soil lysis/GeneRite column purification 0.64 ± 0.25 (11)Boreal soil lysis/Aurora purification 0.78 ± 0.09 (5)Bench ESP lysis/SPE purification 0.0 ± 0.0 (5)c

Bench ESP lysis/GeneRite column purification 0.80 ± 0.41 (14)d

Full ESP lysis/GeneRite column purification 0.78 ± 0.17 (4)e

ND = not determined.a Includes desalted samples.b For experiments in which the 1:100 dilution provided signal.c For experiments in which the 1:10 dilution provided signal.d Includes V. parahaemolyticus recovery.e Only for V. parahaemolyticus spiked into PCR-inhibited seawater; E. faecium

values were not determined because diluted samples fell below the lowest standardof the curve. n = 4 separate experiments, representing a total of 12 filters.

A.M. Cox, K.D. Goodwin / Marine Pollution Bulletin 73 (2013) 47–56 53

3.3.1. Purification methods compared with lysis held constantDNA recovery was compared for various purification methods

using the same lysis procedure (GeneRite lysis), but recovery waslow confounding calculation of inhibition factors (Fig. 3a). Recov-ery was highest (p < 0.05) with Qiagen column purification (QIA),higher even than GeneRite purification (GEN) despite that Gene-Rite lysis was used in both cases. Addition of tRNA to lysates didnot improve recovery from columns (Fig. 3a, GEN/t, QIA/t). SPEpurification on the standalone MFB (SPE) was inhibited (IF = 0,Fig. 3a) and Ct values for the undiluted extracts were below thelowest standard making results DNQ (detectable but not quantifi-able); however, when extracts were diluted to overcome inhibi-tion, recovery was within a factor of 3.4 between SPE, Qiagen,and GeneRite purification.

3.3.2. Lysis methods compared with column purification held constantIn one experiment, Qiagen lysis (QIA) provided the highest cop-

ies recovered, although recovery was not significantly different(p < 0.05) from Boreal soil lysis (BOR) or bead beating (BEA)(Fig. 3b). In general, GeneRite purification did not significantly im-prove inhibition factors, although some improvement was ob-served for bead beating (Table 5). Variability with bead beatingand Boreal soil lysis (36%, 34%, respectively) was relatively high(e.g., compared to 4% Qiagen, 8% GeneRite; Fig. 3b).

Me

Cop

ies

reco

vere

d

0.69

0.32

Method

Cop

ies

reco

vere

d

0.43

0.46

0.78

A0.76

Fig. 4. Box and whisker plots comparing the number of copies of enterococci recoveredseawater using various methods. See Table 1 for abbreviations; SCO/44 and SCO/70 = 44 lfactors for individual experiments are given above each box except for cases in which d

3.3.3. Lysis methods paired with electrophoretic purificationIn general, SCODA purification did not improve inhibition fac-

tors (Table 5) or overall DNA recovery (Figs. 2 and 3a), despite avariety of modifications including sample desalting (BOR/AMI/SCO, Fig. 4b), increasing the volume of buffer added to the elutionchamber (SCO/70, Fig. 4c), and variations with agarose gel and run-ning conditions (data not shown). A new version of the SCODAinstrument became available which reduces sample leakage andimproves variability via a disposable, premade gel system (BorealGenomics, personal communication). Samples sent to BorealGenomics for testing on the new Aurora model showed that DNArecovery from the Aurora model was higher, less variable, and lessinhibited compared to the older SCODA system (Fig. 4a, BOR/AURvs. BOR/SCO). The Aurora model provided the most concentratedextract (data not shown), but it did not improve recovery or inhi-bition factor values compared to purification by spin column(Fig. 4a, BOR/AUR vs. BOR/GEN).

3.3.4. Lysis on a full ESP systemLysis on a full ESP differs from the ESP bench method used with

the standalone MFB because the full system exerts pressure on thefilter during lysis, which was hypothesized to improve lysis. PCRinhibited seawater samples spiked with both E. faecium and V.parahaemolyticus were lysed either on the full ESP or by the Gene-Rite kit (Fig. 5A and B). In both cases, lysates were purified byGeneRite columns. Even with the added pressure during lysis,recovery was still greater for GeneRite-lysed samples for E. faeciumand, additionally, V. parahaemolyticus targets (p < 0.05; Gene-Rite:ESP ratio: E. faecium = 80 ± 21, V. parahaemolyticus = 13 ± 2;avg ± std, n = 6 composite lysates, 18 total filters). Inhibition factorscould not be calculated for E. faecium for these experiments butwere similar for V. parahaemolyticus for the two treatments(Table 5). Although lysates from multiple filters were combinedto reduce variability, similar variability was measured for non-composite lysates (ESP-lysis ranged 2–21% CV, GeneRite-lysisranged 4–21%CV compared to Table 4).

4. Discussion

The ultimate number of targets delivered to a qPCR reaction de-pends on: (1) DNA extraction efficiency, (2) concentration of ex-tract, (3) amount of extract used in the qPCR reaction, (4)inhibition, and (5) the ability to overcome inhibition. This combi-nation of factors can lead to erroneous quantification or false neg-ative results; particularly for low numbers of fecal indicatorbacteria and bacterial pathogens in environmental samples. How-ever, reproducible and accurate DNA recovery is important formicrobial water quality assessments, especially as molecularmethods move into a regulatory framework for rapid bacterialdetection and quantitative MST, and as work moves forward on

thod

0.77

0.56

B

Method

Cop

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reco

vere

d

0.79

0.45

C0.54

from undiluted DNA extracted from filters (A and B) or pellets (C) of concentratedL and 70 lL of buffer added to SCODA chamber, respectively. The average inhibitionilution lowered the Ct value below that of the lowest standard.

Method

Cop

ies

reco

vere

d0.96

A0.85

Method

Cop

ies

reco

vere

d

0.86

1.23

0.70

B0.96

Fig. 5. Box and whisker plots comparing lysis on the full ESP to GeneRite. Graphs show the number of copies recovered from filtered samples of seawater inhibited withsediment and spiked with (A) E. faecium and (B) V. parahaemolyticus (n = 3 composite lysates, representing a total of 9 filters). Filtered samples were lysed either on the fullESP (ESP.1, ESP.2) or by GeneRite kit (GEN.1, GEN.2). All samples were purified via GeneRite kit and run by bench-top qPCR. The value above each box is the inhibition factor(IF) with values greater than 0.81 indicating a lack of inhibition. For ESP trials, IF values were not calculated for E. faecium because diluted samples fell below the loweststandard of the curve.

54 A.M. Cox, K.D. Goodwin / Marine Pollution Bulletin 73 (2013) 47–56

automated detection systems such as the ESP (Stewart et al., 2008;Stoeckel et al., 2009; US EPA, 2010a).

4.1. Recovery

Results differed greatly based on sample (culture spiked PBS vs.PCR-inhibited seawater). While the Boström method produced thehighest recovery with culture spiked PBS (Fig. 1), the GeneRite kit,which uses bead beating, produced the highest recovery with PCR-inhibited seawater (Fig. 2a). The Boström method uses an alcoholprecipitation, which in this study for PCR-inhibited seawater, theprecipitate was observed to be brown. This has been noted beforein soil studies and it is thought that alcohol precipitation co-pre-cipitates humic acids (Ogram et al., 1987).

The Boreal soil lysis method improved recovery of GeneRite andQiagen kits (Figs. 3 and 4; BOR, BOR/GEN, and BOR/QIA). Thismethod was particularly aggressive, employing proteinase K, SDS,CTAB, bead beating, and heat. The importance of sample lysis alsowas suggested with the experiments performed in association withthe full ESP in which better relative recovery was observed for aGram negative bacterium (V. parahaemolyticus) compared to aGram positive (E. faecium). These data suggest that PCR quantifica-tion could benefit from more aggressive lysis of samples.

The ESP is still being optimized for microbial water quality tar-gets (MBARI, personal communication), which may require hard-ware engineering. SCODA was expected to provide good recoverywith concentrated seawater samples because of previous workpurifying inhibited samples (Broemeling et al., 2008). Therefore,the SCODA technology was assessed to see whether it might offerbenefits to in situ sensing. Performance compared to other meth-ods (Fig. 4) coupled with cost and time considerations suggestedthat further exploration of such technology for the ESP was notwarranted at this time. It is unclear why SCODA did not show supe-rior DNA recovery for the seawater samples tested here. Perhapsrecovery was compromised by incomplete lysis, loss during desalt-ing, or DNA may have adhered to particles that were removed dur-ing SCODA purification. SCODA may work to concentrate smallamounts of DNA from dirty samples (Broemeling et al., 2008),whereas, the goal here was to maximize both DNA yield and purity.

A full comparison of lysis reagents could not be made becausemany were proprietary. However, most lysis buffers contain simi-lar basic ingredients – buffers and salts to stabilize pH, detergent todisrupt cell membranes, and chelating agents to help deactivateDNAases. Some of the lysis buffers were known to contain a guan-idinium salt to denature proteins (ESP bench lysis, Qiagen, Mo Bio),and the GeneRite column is compatible with guanidinium salts(GeneRite, personal communication). Therefore, observed differ-ences between protocols (Fig. 3A) could not be explained by anabsence of chaotrophic salts alone.

4.2. Variability

The variability observed in this study (e.g., Table 4) indicatesthat reliable and standardized methods are needed to assess andcorrect for DNA recovery. Variable recovery was observed in thisstudy even with samples spiked with culture (Figs. 1 and 5). Highvariability was seen despite replicate lysates being compositedfrom multiple filters (up to 21% CV even for a bench protocol). Evenwith the normalization procedures used in the EPA protocol forqPCR of enterococci (US EPA, 2010a), the%CV can run �10% (Hau-gland et al., 2005; Shanks et al., 2011), and higher variability hasbeen observed for samples purported to be inhibited (Shankset al., 2011).

4.3. Inhibition

Overall, these data suggest that inhibition removal and recoveryare a tradeoff, and better methods to remove inhibitors are needed.For example, the Boreal Soil method of lysis with GeneRite columnpurification (Fig. 3b, BOR) provided more recovery than the Gene-Rite procedure alone (Fig. 3b, GEN) (perhaps due to improved lysis,see Section 4.1), but the average IF was 0.64 compared to 0.85 forthe GeneRite kit alone (Table 5). In another example, MoBio kitsproduced DNA free from PCR inhibition (IF = 0.80, Fig. 2a, MOB)compared to bead beating (BEA, IF = 0.32), but recovery was rela-tively poor. This tradeoff has been indicated in other inhibitor re-moval studies, where it was observed that DNA recovery wasreduced when a step was added (Bourke et al., 1999; Roose-Amsa-leg et al., 2001; Schriewer et al., 2011).

A dilution method to account for inhibition was used in thisstudy rather than a dilution with spike method (Cao et al., 2012)because the capacity to spike was not available on the ESP orMFB, and spiking may pose a contamination risk. However, dilu-tion does not provide a universal remedy for inhibition becauseit can render the sample too dilute for quantification (Fig. 5). Otherprocedures to provide inhibition controls, such as those describedfor enterococci qPCR (US EPA, 2010a; Haugland et al., 2005; Shankset al., 2011; Haugland et al., 2012) add significant labor and ex-pense, and such standardized procedures for MST studies haveyet to be described (Boehm et al., in press; Stewart et al., in press).

It should be noted that the seawater matrix is complex and likelyincludes a variety of inhibitors (Opel et al., 2010) that could requirea combination of approaches to treat. Besides humic acids, soil alsocontains inhibitors such as fulvic acids, polysaccharides, and metalions (Kermekchiev et al., 2009; Watson and Blackwell, 2000; Yeateset al., 1998), and these are likely to be present in seawater samplesdue to coastal flows. Approaches exist to treat inhibition during thePCR reaction itself, such as BSA, T4 gene 32 protein, or

A.M. Cox, K.D. Goodwin / Marine Pollution Bulletin 73 (2013) 47–56 55

environmental master mixes (Kreader, 1996; Schriewer et al., 2011;Cao et al., 2012), but these were not tested in this study.

4.4. Overall sample preparation and quantification

Sample preparation requirements will increase as the demandto apply quantitative molecular methods grows in food, medical,and environmental industries. Variable extraction efficiency andthe trade-off between DNA quantity and quality (Leff et al., 1995;Lemarchand et al., 2005) is not problematic in presence/absenceanalysis as long as minimum detection limits are met. For qPCR,these issues must be addressed in a quantitative manner, becausedifferent results will be obtained if inhibition is not accounted for(Table 4). Typical strategies utilize a complex set of controls to ad-dress inhibition and recovery (US EPA, 2010a, Cao et al., 2012), andsubstantial debate surrounds the best approach. Such strategiesadd significant expense to all qPCR endeavors, and additional logis-tical challenges for automated platforms. It appears that true quan-tification will remain a goal as long as sample preparationimpediments remain. In the long run, the solution may be thatqPCR will be replaced by an alternative technology, such as dropletdigital PCR. Not only is droplet digital PCR more resistant to PCRinhibition, it eliminates the need for references materials or stan-dards (Morisset et al., 2013), which are significant barriers to theutilization of qPCR in water quality applications (Gooch-Mooreet al., 2011; Cao et al., 2013).

5. Conclusions

Sample preparation methods significantly affected qPCR interms of the copies of target recovered, variability, and the degreeof sample inhibition. Results differed for samples of culture vs. sea-water samples that caused PCR inhibition, indicating the need toassess method performance with challenging samples. In general,lysis that included bead beating and spin column purification pro-vided good relative recovery and inhibitor removal. Nonetheless,the best performing kit in this study (GeneRite) was improved bymodification to a more aggressive lysis protocol (the Boreal soilprotocol). Both the lysis and purification steps employed on thecurrent ESP performed poorly relative to other tested methods,suggesting that engineering modifications to improve samplepreparation on the ESP are warranted. However, relative perfor-mance of SCODA purification technology did not appear to warrantfurther exploration for the ESP. Overall, differential recovery andinhibitor removal was observed, highlighting the need for reliablediagnosis of sample loss and inhibition for quantitative PCR appli-cations, including automated biosensors. In general, this is an areaof research that requires further development.

Acknowledgements

The Southern California Coastal Water Research Project(SCCWRP) is gratefully acknowledged for use of their standaloneMFB/SPE module equipped with qPCR. The NOAA Northwest Fish-eries Science Center (NWFSC) graciously allowed access to theirfull ESP for laboratory experiments. Particular recognition is givento Blythe Layton (SCCWRP) for assistance and training with thestand-alone MFB and to William Nilsson and Mark Strom (NWFS)for assistance on the full ESP. Appreciation is given to SpyglassInc. staff for training on the ESP system. Thanks to D. Lim and S.Leskinen (University of South Florida) for providing water samplesfrom the PMACS 1000TM. Appreciation is given to the F. Azam lab-oratory (Scripps Institute of Oceanography) for access to micros-copy facilities. Thanks to D. Broemeling, J. Pel, and A. Marzali(Boreal Genomics) for assistance with SCODA optimization and

for running experiments on the Aurora instrument. A. Cox was sup-ported through the NOAA Oceans and Human Health Initiative andthe National Research Associateship Program.

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