Comparative evaluation of automateextraction methods for detection ofrom suspensions and spiked sw

chain reactiLeslie A. Dauphina,, Roblena E. Walkera, Jea

aBioterrorism Rapid Response and Advanced Technology (BRRAT) Lab

Available online at www.sciencedirect.com

Diagnostic Microbiology and Infectious DKeywords: Francisella tularensis; DNA extraction; Tularemia; Real-time PCR; Bioterrorism

1. Introduction

Francisella tularensis is a Gram-negative, nonmotile,aerobic coccobacillus that is the causative agent of tularemia,a zoonotic infection with a broad host distribution. Currently,there are 3 recognized subspecies, tularensis, holarctica, andmediasiatica, which differ with regard to their biochemicalproperties and geographical distribution (Keim et al., 2007;Oyston, 2008). F. tularensis subsp. tularensis (type A) and

Disclaimer statement: The findings and conclusions in this report arethose of the author(s) and do not necessarily represent the official position of theCenters for Disease Control and Prevention/the Agency for Toxic Substancesand Disease Registry. Names of vendors or manufacturers are provided asexamples of available product sources; inclusion does not imply endorsementof the vendors, manufacturers, or products by the Centers for Disease Controland Prevention or the US Department of Health and Human Services. Francisella tularensis is a select agent, and its possession, use, and

transfer is regulated by the US Department of Health and Human Services,Centers for Disease Control and Prevention. The select agent regulationsAbstract

This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitablefor real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automatedmethods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, theMasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit.The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases oftularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis atconcentrations of 106 CFU/mL. Real-time PCR analysis using a multitarget 5 nuclease assay for F. tularensis revealed that the PCRsensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samplesthan the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection ofF. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.Published by Elsevier Inc.Division of Preparedness and Emerging Infections (DPEI), Centers for Disease Control and Prevention (CDC), Atlanta, GA 30333, USAbDiagnostic and Reference Laboratory, Bacterial Diseases Branch, Division of Vector-Borne Diseases, CDC, Fort Collins, CO 80521, USA

Received 25 May 2010; accepted 28 February 2011have mandatory reporting requirements for identification of select agents indiagnostic specimens.

Corresponding author. Tel.: +1-404-639-4991; fax: +1-404-639-4234.E-mail address: ldauphin@cdc.gov (L.A. Dauphin).1 Present address: Gastroenteritis and Respiratory Viruses Laboratory

Branch, Division of Viral Diseases, CDC, Atlanta, GA 30333, USA.

0732-8893/$ see front matter. Published by Elsevier Inc.doi:10.1016/j.diagmicrobio.2011.02.010d and manual commercial DNAf Francisella tularensis DNAabs by real-time polymeraseon,

nnine M. Petersenb, Michael D. Bowena,1

oratory, Laboratory Preparedness and Response Branch (LPRB),

isease 70 (2011) 299306www.elsevier.com/locate/diagmicrobioholarctica (type B) cause the majority of reported cases ofdisease in humans, with subsp. tularensis causing the moresevere disease (Ellis et al., 2002). Molecular subtyping hasfurther separated F. tularensis subsp. tularensis into 2genetically distinct clades, A.I and A.II, which display

differences in transmission, disease outcome, and geograph-ic distribution (Farlow et al., 2005; Johansson et al., 2004;Staples et al., 2006) F. tularensis strain SCHU S4 is theproposed subsp. tularensis-type strain (Ellis et al., 2002).

F. tularensis is highly infectious, which has led manynations including the USA, the former Soviet Union, andJapan to regard it as a potential biological weapon (Denniset al., 2001). Diagnostic methods for the identification of

for biothreat agents. Furthermore, the aforementioned studycompared manual DNA extraction kits, which do not providethe high-throughput capacity that may be required inlaboratories that process large numbers of specimens.

The purpose of this study was to evaluate commercialautomated and manual extraction methods for the recoveryof F. tularensis DNA. Since environmental samples areamong the most common sample types submitted for testing

F. tularensis cultures were initiated from lyophilized

cessio

987,418,387, O

et ale Div

300 L.A. Dauphin et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 299306F. tularensis include culturing with cysteine-enrichedmedia and testing by fluorescent-labeled antibodies; how-ever, these methods are either time consuming, not useful fordiagnosis of acute infections, or lacking in sensitivity(Versage et al., 2003). The potential for F. tularensis as abiological weapon has highlighted the need for rapiddiagnostics; hence, several real-time PCR assays have beendeveloped (Kugeler et al., 2006; Mitchell et al., 2010; Molinset al., 2009; Tomaso et al., 2007; Versage et al., 2003). Thesetypes of assays are used by select laboratories of theLaboratory Response Network (LRN), a diagnostic labora-tory network for potential bioterrorism threats, for thepresumptive identification of F. tularensis in clinical andenvironmental specimens (Rotz & Hughes, 2004).

As the use of molecular diagnostics, such as rapid real-timePCR assays, has become routine in diagnostic laboratories,specimen throughput has increased accordingly (Schuurmanet al., 2007). Manual nucleic acid extraction methods have thepotential to be the rate-limiting component of rapid testingprotocols because of their limited throughput and laborintensiveness (Knepp et al., 2003). Automated methods canbe a viable alternative to manual methods because they offermedium- to high-throughput options for nucleic acidextraction. It has been widely reported that the efficiencyof DNA extraction can influence the subsequent sensitivityof PCR assays (Black & Foarde, 2007; Dauphin et al., 2009;Durnez et al., 2009; Queipo-Ortuno et al., 2008; Schuurmanet al., 2005); therefore, the selection and use of optimalextraction methods are crucial for laboratory diagnostics.

Few previous studies have evaluated DNA extractionmethods specifically for the isolation of F. tularensis DNA.One study (Whitehouse & Hottel, 2007) compared commer-cial DNA extraction methods for the recovery of F. tularensisDNA and reported that the UltraClean Microbial DNAisolation kit (MoBio Laboratories, Carlsbad, CA) and thePowerMax kit (MoBio Laboratories) were optimal amongthose studied. This study (Whitehouse & Hottel, 2007) usedsoil samples, which is an important sample type, but is onlyone of the types submitted to laboratories that perform testing

Table 1Francisella tularensis strains used in this studya

Subspecies (type) CDC ac

F. tularensis subsp. tularensis (A.I) MA00-2F. tularensis subsp. tularensis (A.II) WY96-3F. tularensis subsp. holarctica (B) KY99-3

a Strain information from Molins-Schneekloth et al. (2008) and Versageb CDC accession number, excluding SCHU S4 and LVS, assigned by thstocks, which were reconstituted with 0.5 mL of Brain HeartInfusion broth. The cultures were streaked for isolatedcolonies onto cysteine heart agar supplemented with 9%sheep blood (CHAB) plates, and the plates were incubatedfor 48 h at 37 C. For each strain, a single colony wastransferred to 1 mL of sterile physiologic saline (0.85%sodium chloride) using a sterile inoculating loop and mixedby vortexing at low speed for 30 s. A 200-L aliquot of each

n numberb Origin

SCHU S4 Massachusetts, OhioNM99-1823 Wyoming, New MexicoR96-0246, LVS Kentucky, Oregon, Russia

. (2003).ision of Vector-Borne Diseases, CDC, Fort Collins, CO.during suspected biothreat situations (Luna et al., 2003), andculture is used for confirmatory testing, this study compared6 methods, including 2 automated methods and 4 manualmethods, using bacterial suspensions and spiked swabs. Theevaluation included viability testing of the DNA extracts byculture, limit of detection (LOD) studies for phosphate-buffered saline (PBS) suspensions and spiked swab samples,and assessments of DNA yields and DNA purity.

2. Materials and methods

2.1. Biosafety procedures

All procedures using F. tularensis strains were performedin a biosafety level 3 laboratory using relevant containmentand safety precautions, including the use of a powered air-purifying respirator.

2.2. F. tularensis strains and culture

The 7 F. tularensis strains used in this study (Table 1) wereprovided by the Division of Vector-Borne Diseases, CDC(Fort Collins, CO) and have been described in previousreports (Molins-Schneekloth et al., 2008; Versage et al.,2003). Six of the 7 strains were selected as representative ofthe 2 F. tularensis subsp. which are clinically relevant, subsp.tularensis (strains MA00-2987, SCHU S4, WY96-3418, andNM99-1823) and subsp. holarctica (strains KY99-3387and OR96-0246). The live-vaccine strain (LVS) was usedas a positive-control strain for real-time PCR assays.

301L.A. Dauphin et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 299306suspension was then spread onto CHAB plates in triplicateand the plates were incubated for 48 h at 37 C. Cultureswere harvested into 10 mL of sterile PBS (0.01 mmol/L, pH7.4) using sterile Dacron fiber-tipped swabs (FisherScientific, Pittsburgh, PA), which were pre-moistened withPBS. To determine the bacterial concentration, serial 10-folddilutions of this suspension were performed in physiologicsaline, 100-L aliquots were spread onto CHAB plates intriplicate, and the plates were incubated as described above.Colonies were counted using standard microbiologicalmethods, and the quantified F. tularensis suspensions werestored at 70 C until use.

2.3. Spiking of swabs

Three types of commonly used swabs were used for thespiking experiments, cotton (Fisher Scientific), polyester(Fisher), and foam (Epicentre Biotechnologies, Madison,WI). Tenfold serial dilutions of F. tularensis strain SCHUS4 with a starting concentration of 107 colony forming units(CFU)/mL were performed in PBS, and the swabs wereinoculated in triplicate with 10-L aliquots at eachconcentration. The swabs were allowed to air dry for 30min at room temperature and were then processed forF. tularensis recovery using the Swab Extraction TubeSystem (SETS) (Roche Applied Sciences, Indianapolis, IN).The SETS is a disposable centrifugal system consisting of aninner tube (with a hole) and an outer collection tube, and itsuse for the recovery of F. tularensis from swabs has beendescribed in detail previously (Walker et al., 2010). Therecovered F. tularensis PBS suspensions were immediatelyused for the DNA extraction procedures.

2.4. Automated and manual DNA extraction methods

Six commercial DNA extraction methods, including 2automated instruments and 4 manual kits, were evaluated inthis study. All of the procedures were performed in triplicateusing 200-L aliquots of F. tularensis as either quantifiedPBS suspensions or samples recovered from spiked swabs.Following the DNA extraction procedures, the PCR-readyDNA samples were stored at 20 C until use.

The 2 automated DNA extraction methods used differentprinciples for DNA extraction. The MagNA Pure Compactinstrument (Roche) utilized magnetic bead technology,whereas the QIAcube (Qiagen, Valencia, CA) employed asilica spin-filter (column) technology. For the MagNA PureCompact instrument, DNA extractions were performed usingthe MagNA Pure Compact Nucleic Acid Isolation Kit I(Roche). As a safety precaution, an external lysis protocolwas performed by combining each sample with 300 L ofMagNA Pure LC DNA Isolation Kit I Lysis/Binding Buffer,followed by incubation at room temperature for 30 min.Immediately following the lysis procedure, the samplesunderwent automated extraction on the MagNA PureCompact instrument using the DNA Blood External LysisPurification Protocol, with an elution volume of 100 L. Forthe QIAcube instrument, DNA extractions were performedusing the QIAamp DNA Blood Mini Kit (Qiagen).Automated DNA extraction on the QIAcube was performedusing the Blood and Body Fluid Spin Protocol with anelution volume of 200 L.

The 4 manual DNA extraction kits used 3 differentprinciples for DNA extraction. Both the IT 1-2-3 DNASample Purification Kit (Idaho Technology, Salt Lake City,UT) and the UltraClean Microbial DNA Isolation Kit(MoBio Laboratories) combine bead-beating and spin-column technologies. The MasterPure Complete DNA andRNA Purification Kit (Epicentre) uses a precipitationmethodology, whereas the QIAamp DNA Blood Mini Kit(Qiagen) utilizes the same silica spin-filter technology asnoted above. All of the manual DNA extraction methodswere performed according to the manufacturer's instructions.These procedures have been described in detail previously(Dauphin et al., 2010).

2.5. Viability testing of DNA extracts

To assess each extraction method's ability to inactivateF. tularensis strains, the DNA extracts were tested for bacterialviability. A total of 126 DNA samples were tested, whichincluded the following for each of the 6 DNA extractionmethods evaluated: triplicate sample extracts from F. tular-ensis strain SCHU S4 at a concentration of 106 CFU/mL (18samples), and triplicate sample extracts from 6 of theF. tularensis strains listed in Table 1 (MA00-2987, SCHUS4, WY96-3418, NM99-1823, KY99-3387, and OR96-0246)at a concentration of 105 CFU/mL (108 samples). Immediatelyfollowing each extraction procedure, 10% of the volume ofeach sample extract was spread onto CHAB plates and theplates were incubated for up to 5 days at 37 C. As a control forthis viability testing, an equal volume of each stock livebacterial suspension was spread onto CHAB plates and theplates were incubated as described above. Viability wasdetermined by observation of the plates for colonies. As anadditional safety precaution, the remaining volume of theviability-tested extracts, as well as all other DNA extractsprepared in this study, was micro-filtered using 0.1-mcentrifugal filter units (Millipore, Billerica, MA) using amethod described previously for removal ofBacillus anthracisspores from DNA preparations (Dauphin & Bowen, 2009).

2.6. DNA yield and purity

DNA extracted from 3 F. tularensis strains, including 1representative each for type A.I (SCHU S4), type A.II(NM99-1823), and type B (KY99-3387), was quantified froma concentration of bacteria at 106 CFU/mL using a NanoDrop8000 Spectrophotometer (ND Technologies, Wilmington,DE). The spectrophotometer was blanked before measure-ment with the corresponding elution buffer for each DNAextraction method. For the MagNA Pure Compact, thespectrophotometer was blanked with the elution buffer for theMagNA Pure LC extraction kit. For each sample, the

3 replicates produced a positive result for all 3 real-time PCRtargets, as indicated by C values of 40. To compare the

302 L.A. Dauphin et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 299306absorbance at 260 nm (A260) was measured. This value wasused to calculate the average concentration of DNA for eachset of triplicate samples by multiplying the A260 measurementby a conversion factor (50 g/mL per 1 A260 unit for double-stranded DNA). To estimate the purity of DNA extracts, theabsorbance at 280 nm (A280) was measured also and theaverage ratio between the A260 nm and A280 nm (A260/A280)was calculated for triplicate samples. Samples with A260/A280ratios between 1.8 and 2.0 were presumed to be free ofsignificant contamination (Manchester, 1995).

2.7. Preparation of positive controls

Cultures of F. tularensis strain LVS were prepared andused as a positive control in real-time PCR assays. Thecultures were harvested into 250 L of sterile deionized waterin microcentrifuge tubes. The samples were vortexed briefly,boiled for 5 min, and pelleted by centrifugation for 30 s at10,000 g. The supernatants were transferred to 0.1-m filterunits and filtered as described previously. Filtered cell lysateswere diluted in Tris-EDTA buffer to dilutions whichproduced real-time PCR cross-threshold (CT) values between25 and 30 using the real-time PCR signature sets describedbelow. The positive-control samples were stored at 20 Cthroughout this study.

2.8. Real-time PCR analysis

The multi-target real-time PCR assay described byVersage et al. (2003) was used to evaluate the 6 DNAextraction methods for the recovery of F. tularensis DNAfrom PBS suspensions and spiked swabs. The assay wasdeveloped for the specific detection of F. tularensis inclinical and environmental specimens by targeting multiplegenetic loci: the ISFtu2 insertion element, the 23kDa gene,and the tul4 gene (Versage et al., 2003). PCR was performedusing 25-L reaction volumes, each of which contained afinal concentration of 1X LightCycler FastStart DNAMasterHybProbes PCR Master Mix (Roche Molecular Biochemi-cals, Indianapolis, IN), 500 nmol/L of each PCR primer, 100nmol/L of each FAM-labeled TaqMan probe, 4 mmol/LMgCl2 (23kDa and tul4 assays) or 5 mmol/L MgCl2 (ISFtu2assay), 0.5 U of uracil-DNA glycosylase (Roche), and 5 Lof either DNA extract, positive control DNA, or water (forno-template controls). As an additional control, an internal-positive-control (IPC) real-time PCR assay (Applied Bio-systems, Foster City, CA) was used to assess the ability ofeach DNA extraction method to remove potential PCRinhibitors. The IPC reagents included a control DNA, PCRprimers, and a VIC-labeled hydrolysis probe. These reagentswere added to each PCR reaction and were run in thepresence of each DNA extract.

Real-time PCR was performed on the 7500 Fast Real-Time PCR System (Applied Biosystems) using the standard7500 operational setting and a thermocycling profileconsisting of an incubation at 50 C for 2 min (glycosylasestep), a hot-start Taq activation step of 95 C for 10 min,T

extraction methods for the recovery of DNA amongF. tularensis strains, real-time PCR was performed usingtriplicate sample extracts prepared from 6 strains ofF. tularensis at a concentration of 105 CFU/mL. Compar-isons for the isolation of F. tularensis DNA from swabspecimens were performed using triplicate DNA extractsprepared from swabs spiked with dilutions of F. tularensisstrain SCHU S4. The LOD for spiked swabs was determinedas described above. All PCR runs were repeated 2 times for atotal of 3 PCR runs for each comparative evaluation.

2.9. Statistical analysis

To determine whether the variability of CT values forF. tularensisDNAextracted fromPBS suspensions and spikedswab samples was significant, the mean CT values for eachmethod were compared using 1-way ANOVA (n = 27). Thisanalysis included data for triplicate sample extracts from strainSCHU S4, which was tested in a 3-target real-time PCR assayfor a total of 3 PCR runs (9 data points per PCR run for eachextraction method). To determine whether the strain-to-strainvariability ofCT values for DNA extracted from6F. tularensisstrains was significant, the CT values were compared using1-way ANOVA (n = 54). This analysis included data fortriplicate sample extracts from the 6 strains, which were testedin a single target real-time PCR assay for a total of 3 PCRruns (18 data points per PCR run for each extraction method).When significant differences were identified, Tukey'smultiple comparison test was used to perform nonparametricpair-wise analyses of the CT values.

3. Results

3.1. Evaluation of DNA extraction methods for inactivationof F. tularensis strains

All of the DNA extraction methods were highly efficientat killing all F. tularensis strains at concentrations up to 106

CFU/mL as there was no growth observed in cultures of suchDNA extracts prepared using each of the 6 methods. Theviability testing controls were positive for each strain ofF. tularensis tested. Since 10% of the volume of each DNAextract was used for viability testing, this would indicate atfollowed by 45 cycles of 95 C for 10 s and 60 C for 30 s,and then 45 C for 5 min. Data collection and analysis wereperformed using the 7500 Fast System Sequence DetectionSoftware, version 1.4, with the 21 CFR Part 11 electronicrecords module.

To compare the automated and manual DNA extractionmethods for the recovery of DNA from F. tularensis, weperformed real-time PCR using triplicate DNA extractsprepared from F. tularensis strain SCHU S4 at concentra-tions ranging from 106 to 100 CFU/mL. The LOD wasdetermined to be the lowest concentration for which 3 out of

NA extraction methods for their efficiency of DNAxtraction from 6 different strains of F. tularensis. Fig. 1hows the range of CT values for the ISFtu2 gene target frome 6 strains at a concentration of 105 CFU/mL. TheasterPure kit yielded DNA having the lowest average CTalues (equating to the best quality/quantity), with a triplicateample range (mean) of 19.725.9 (22.9), followed by theIAcube and the MagNA Pure Compact, with CT valueanges (mean) of 20.826.5 (23.6) and 20.726.4 (23.9),espectively. DNA extraction using the QIAamp kit and theltraClean kit resulted in CT values with ranges (mean) of0.927.2 (24.1) and 24.031.6 (27.2), respectively. The IT 1--3 kit yielded DNA with the highest CT values (28.437.3)nd a mean CT value of 32.5. The differences in mean CTalues for the 6 DNA extraction methods were found to beignificant by 1-way ANOVA (P = 0.0001; n = 54). Pair-wiseomparisons of CT values indicated significant differencesetween theUltraClean kit and the IT 1-2-3 kit when comparedthe 4 other DNA extraction methods (P b 0.05; n = 54).

.4. Comparison of extraction methods for DNA yieldnd purity

Table 3 shows the DNA extract volumes, average DNAoncentrations, yields, and the range of A260/A280 ratios for

303L.A. Dauphin et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 299306least a 5-log reduction of viability for the 6 DNA extractionmethods evaluated in this study.

3.2. Evaluation of DNA extraction methods by real-time PCR

Table 2 shows the LOD by real-time PCR for DNAextracted from F. tularensis strain SCHU S4 at concentra-tions ranging from 106 to 100 CFU/mL with the 6 DNAextraction methods. DNA extraction using the MagNA Pure

Table 2Real-time PCR LOD for DNA isolated from F. tularensis using automatedand manual DNA extraction methods

Extractionmethoda

LOD(CFU/mL)b

Average CT (mean SD)c,d

ISFtu2 23kDa tul4

MagNA PureCompact

103 29.6 1.35 32.8 0.73 33.0 0.30

QIAcube 103 31.0 0.27 34.8 0.65 34.5 0.65IT 1-2-3 105 32.5 1.42 32.3 1.64 34.9 0.85MasterPure 103 29.3 0.29 31.8 0.96 32.0 0.72QIAamp 103 30.8 0.60 33.6 1.12 36.1 3.65UltraClean 104 29.0 1.16 32.9 1.15 33.0 2.14

a Extraction methods were performed in triplicate using F. tularensisstrain SCHU S4 at concentrations ranging from 106 to 100 CFU/mL.

b The LOD was determined to be the lowest concentration for which 3out of 3 replicates produced a positive result for the 3 real-time PCR targetsas described by Versage et al. (2003).

c The mean CT values (at the determined LOD) are shown todemonstrate levels of reproducibility among replicate sample DNA extracts.

d The differences in mean CT values between the 6 methods were foundto be significant by 1-way ANOVA (P = 0.001; n = 27). Pair-wisecomparisons using Tukey's multiple comparison test revealed significantdifferences between the IT 1-2-3 kit and the UltraClean kit and each of theother 4 extraction methods (P b 0.05; n = 27).Compact, the QIAcube, the MasterPure kit, and the QIAampkit yielded the most sensitive levels of detection by real-timePCR, followed by the UltraClean kit, with the IT 1-2-3 kityielding DNA with the poorest level of detection. The LODwas 103 (CFU/mL in the original cell suspension) for theDNA extracted with the 4 methods which performed thebest. Since a 5-L volume of DNA extract was used for PCRreactions, this would translate to 5 CFU per PCR reaction forthese 4 methods. The UltraClean kit yielded DNA whichresulted in a LOD of 104 CFU/mL (50 CFU/reaction), andthe PCR LOD for DNA prepared with the IT 1-2-3 kit was105 CFU/mL (500 CFU/reaction). The differences in meanCT values for the 6 DNA extraction methods were found tobe significant by 1-way ANOVA (P = 0.001; n = 27). Pair-wise comparisons of CT values indicated significantdifferences between the UltraClean kit and the IT 1-2-3kit, when compared to the 4 other DNA extraction methods(P b 0.05; n = 27).

3.3. Real-time PCR analysis of DNA extracted from 6strains of F. tularensis

Using the real-time PCR results obtained with the indexstrain, SCHU S4, as the baseline LOD, we evaluated the 6the DNA extracted from 3 F. tularensis strains (SCHU S4,NM99-1823, KY99-3387) at a bacterial concentration of 106

CFU/mL. The volumes for DNA extracts ranged from 50 to200 L, with the MasterPure kit producing the smallest DNAvolume and the QIAcube and the QIAamp kit providing the

MPC QC IT MP Q UC10

20

30

40

Extraction Method

Ave

rage

CT

Fig. 1. Box-and-whisker plots showing the distribution of CT valuesobtained using DNA prepared from 6 strains of F. tularensis atconcentrations of 105 CFU/mL using 2 automated DNA extraction methods,the MagNA Pure Compact (MPC) and the QIAcube (QC), and 4 manualDNA extraction methods, the IT 1-2-3 kit (IT), the MasterPure kit (MP), theQIAamp kit (Q), and the UltraClean kit (UC). The middle line for each boxcorresponds to the mean for the observed values. The end points of thewhiskers show the 2.5 and 97.5 percentiles. The differences in mean CTvalues between the 6 DNA extraction methods were found to be significantby 1-way ANOVA (P b 0.0001; n = 54). Tukey's multiple comparison testrevealed significant differences between the mean CT values for theUltraClean kit and the IT 1-2-3 kit, when compared to the 4 other DNAextraction kits (P b 0.05; n = 54).DesthMvsQrrU22avscbto

3a

c

here was no evidence of PCR inhibition for any of the DNAxtraction methods in samples recovered from the 3 swabaterials, as measured by the IPC assay (data not shown).

. Discussion

The increasing availability of commercial DNA extrac-on methods emphasizes the need for a timely evaluation ofepresentative DNA extraction technologies with a range ofample throughput capacities. This study demonstrated thate sensitivity of F. tularensis detection by real-time PCRas equivalent after DNA extraction by both automatedethods tested and by the MasterPure and the QIAampanual methods. Several studies have reported improvedCR sensitivity using DNA extraction kits such as theasterPure kit, the QIAamp kit, and the UltraClean kit

Dauphin et al., 2010; Knepp et al., 2003; Queipo-Ortuno

304 L.A. Dauphin et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 299306greatest volumes. The MasterPure kit yielded DNA with thehighest DNA concentrations for all F. tularensis strains. TheUltraClean kit yielded DNA at the second-highest concen-tration. The MagNA Pure Compact ranked third, followedby the QIAamp kit and the QIAcube instrument. The IT 1-2-3 kit yielded the lowest concentration of DNA. Theautomated QIAcube and the manual QIAamp kit resultedin the greatest total DNA yields , followed by the MagNAPure Compact. The UltraClean kit resulted in the lowest totalDNA yield. The MasterPure, QIAamp, and UltraClean kitsproduced DNA with the highest purity, with triplicate A260/A280 ratios ranging from 1.69 to 1.75, 1.65 to 1.99, and 1.70

Table 3Comparison of sample volume, DNA concentration, yield, and puritybetween automated and manual DNA extraction methods

Extractionmethod

Volume(L)

Concentration(ng/L)

Totalyield (g)a

A260/A280ratiosb

MagNAPure Compact

100 16.10 1.61 1.561.79

QIAcube 200 10.68 2.14 1.621.89IT 1-2-3 200 6.94 1.39 1.261.48MasterPure 50c 23.22 1.16 1.691.75QIAamp 200 11.14 2.23 1.651.99UltraClean 50 19.64 0.98 1.701.88

a Total DNA yields were calculated from mean A260 measurements fortriplicate sample extracts from F. tularensis strains SCHU S4, NM99-1823,and KY99-3387 at an input concentration of 106 CFU/mL, multiplied by theconversion factor, multiplied by the DNA elution/resuspension volume.A 2-L sample was used to quantify the DNA from each extract.

b The range of mean A260/A280 ratios was determined using triplicatesample extracts from F. tularensis strain SCHU S4 at concentrations rangingfrom 106 to 100 CFU/mL.

c The elution volume for this kit was increased from 35 L(manufacturer's recommendation) to 50 L to obtain replicates sufficientfor statistical analyses.to 1.88, respectively.

3.5. Real-time PCR analysis of F. tularensis DNA recoveredfrom spiked swabs

Table 4 shows the real-time PCR LOD for DNA extractedfrom spiked swabs. The 6 extraction methods resulted invarying levels of detection among the 3 swab materials. Forcotton swabs, the MagNA Pure Compact and the QIAampkit performed best, with a LOD of 104 CFU/mL (100 CFU/swab). The QIAcube, the MasterPure kit, and the UltraCleankit ranked second for DNA extraction from cotton swabs,each yielding DNA with a LOD of 105 CFU/mL (103 CFU/swab). The IT 1-2-3 kit yielded DNA with the poorest levelof detection (107 CFU/mL, 105 CFU/swab). For foam swabs,all of the DNA extraction methods resulted in a LOD of105 CFU/mL (103 CFU/swab), with the exception of the IT1-2-3 kit, which resulted in the poorest level of detection(106 CFU/mL, 104 CFU/swab). Likewise, for polyesterswabs, all of the DNA extraction methods resulted in a LODof 105 CFU/mL (103 CFU/swab), except the IT-1-2-3 kit,which produced a LOD of 107 CFU/mL (105 CFU/swab).

swabs spiked with 10-fold dilutions of F. tularensis strain SCHU S4 at astarting concentration of 107 CFU/mL (105 CFU/swab).b The LOD was determined to be the lowest concentration for which 3out of 3 replicates produced a positive result for each of the 3 real-time PCRtargets in the assay described by Versage et al. (2003).

c CFU/mL (CFU/swab).et al., 2008). The results of F. tularensis real-time PCR werecomparable for the MagNA Pure Compact and the QIAcubeinstruments and the MasterPure and QIAamp kits because allof these methods effectively extracted F. tularensis atconcentrations as low as 103 CFU/mL. Furthermore, the 2automated methods resulted in significantly better real-timePCR detection levels when compared to the IT 1-2-3 kit andthe UltraClean kit (Table 2). These results indicate that eitherof the 2 automated DNA extraction methods is a suitablealternative to the manual DNA extraction methods tested inthis study.

Several factors, such as DNA purity, concentration, anddamage, can influence the sensitivity of real-time PCRassays. The results of this study indicated that DNA purityhad a likely influence on the sensitivity of the real-time PCRassay. All of the methods resulted in comparable A260/A280ratios, with the exception of the IT 1-2-3 kit, which had thepoorest values in comparison, and yielded DNA which wassubsequently detected less efficiently. There was also, as

Table 4Real-time PCR LOD for DNA extracted from spiked swabs using automatedand manual DNA extraction methods

Extraction methoda LODb,c

Cotton swabs Foam swabs Polyester swabs

MagNA Pure Compact 104 (100) 105 (103) 105 (103)QIAcube 105 (103) 105 (103) 105 (103)IT 1-2-3 107 (105) 106 (104) 107 (105)MasterPure 105 (103) 105 (103) 105 (103)QIAamp 104 (100) 105 (103) 105 (103)UltraClean 105 (103) 105 (103) 105 (103)

a Extraction was performed in triplicate on samples recovered fromTem

4

tirsthwmmPM(

305L.A. Dauphin et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 299306expected, an apparent correlation between DNA concentra-tion and PCR sensitivity. With the exception of theUltraClean kit, the DNA extraction methods which resultedin the greatest concentrations also resulted in the bestlevels of detection by real-time PCR. With unique regardto the UltraClean kit, neither DNA purity nor DNA con-centration appeared to correlate with PCR sensitivity forF. tularensis suspensions, as this kit yielded better A260/A280ratios and greater DNA concentrations than most of themethods evaluated in this study, yet the DNA it yielded wasdetected among the worst in comparison (Table 2). Onefactor which may have contributed to the lower PCRsensitivity observed with the UltraClean kit is DNA damage.The lysis procedure for the UltraClean kit employed a10-min bead-beating procedure. Mechanical disruption ofF. tularensis cells during the extraction process may haveresulted in DNA shearing and damage to PCR target regionswhich can adversely affect PCR amplification (Scupham etal., 2007). Furthermore, the instruction manual for this kitoffers an alternative lysis method for less DNA shearing,indicating that the manufacturers are cognizant that thebead-beading procedure may not be as appropriate forsome applications.

The ability of extraction methods to purify nucleic acidand efficiently remove inhibitors from various samplematrices is essential when using molecular diagnosticssuch as PCR assays (Boom et al., 1999; Chan et al., 2008).In this study, the automated and manual DNA extractionmethods were compared using spiked swab samples tosimulate specimens which may be tested in diagnosticlaboratories that perform testing for F. tularensis. The resultsindicated that the 2 automated methods performed as well asor better than the manual extraction methods for the recoveryof quality DNA from swab samples (Table 4). Moreover, theresults suggest that the automated methods were as efficientas the manual methods for yielding DNA free of inhibitorysubstances, as indicated by the IPC assay. Finally, theefficiency of DNA extraction for the 2 automated methodswas not compromised when the methods were evaluated forextraction from different swab materials. Combined, thesedata indicate that the MagNA Pure Compact and theQIAcube methods are as sufficient as the manual DNAextraction methods used in this study for the isolation ofF. tularensis DNA from swabs.

One limitation of this study is that spiked swabs wereused to simulate environmental samples that would beprocessed during laboratory bioterrorism-related investiga-tions. It is not clear whether the results would be equivalentfor field-collected environmental samples. In addition, theswabs were processed for testing shortly after inoculation.Future studies should assess equivalence using environmen-tal swabs obtained from surface sampling and assess theimpact of storage times on pathogen detection levels.

The aim of this study was to compare 2 automated DNAextraction methods to manual methods for extraction ofF. tularensis DNA. The results show that the MagNA PureCompact and the QIAcube instruments perform equivalentlyto each other and to other popular commercially availablemanual methods. These 2 instruments offer diagnosticlaboratories the option to select automated methods for theextraction of F. tularensisDNA with optimal PCR sensitivity.

Acknowledgments

The authors acknowledge Liberty Bost, Rebecca Hutch-ins, and Michael Vick for laboratory assistance. The authorsalso thank Gregory Buzard, Pamela Diaz, and HarveyHolmes for editorial comments.

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306 L.A. Dauphin et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 299306

Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DN...IntroductionMaterials and methodsBiosafety proceduresF. tularensis strains and cultureSpiking of swabsAutomated and manual DNA extraction methodsViability testing of DNA extractsDNA yield and purityPreparation of positive controlsReal-time PCR analysisStatistical analysis

ResultsEvaluation of DNA extraction methods for inactivation of F. tularensis strainsEvaluation of DNA extraction methods by real-time PCRReal-time PCR analysis of DNA extracted from 6 strains of F. tularensisComparison of extraction methods for DNA yield and purityReal-time PCR analysis of F. tularensis DNA recovered from spiked swabs

DiscussionAcknowledgmentsReferences