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Diagnostic Microbiology and Infectious Disease 70 (2011) 299 306


www.elsevier.com/locate/diagmicrobio

Comparative evaluation of automated and manual commercial DNA


extraction methods for detection of Francisella tularensis DNA
from suspensions and spiked swabs by real-time polymerase
chain reaction,
Leslie A. Dauphina,, Roblena E. Walkera , Jeannine M. Petersenb , Michael D. Bowena,1
a

Bioterrorism Rapid Response and Advanced Technology (BRRAT) Laboratory, Laboratory Preparedness and Response Branch (LPRB),
Division of Preparedness and Emerging Infections (DPEI), Centers for Disease Control and Prevention (CDC), Atlanta, GA 30333, USA
b
Diagnostic and Reference Laboratory, Bacterial Diseases Branch, Division of Vector-Borne Diseases, CDC, Fort Collins, CO 80521, USA
Received 25 May 2010; accepted 28 February 2011

Abstract
This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable
for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated
methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the
MasterPure 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 of
tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at
concentrations of 106 CFU/mL. Real-time PCR analysis using a multitarget 5 nuclease assay for F. tularensis revealed that the PCR
sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4
methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples
than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of
F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2
automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.
Published by Elsevier Inc.
Keywords: Francisella tularensis; DNA extraction; Tularemia; Real-time PCR; Bioterrorism

Disclaimer statement: The findings and conclusions in this report are


those of the author(s) and do not necessarily represent the official position of the
Centers for Disease Control and Prevention/the Agency for Toxic Substances
and Disease Registry. Names of vendors or manufacturers are provided as
examples of available product sources; inclusion does not imply endorsement
of the vendors, manufacturers, or products by the Centers for Disease Control
and 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 regulations
have mandatory reporting requirements for identification of select agents in
diagnostic 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.2011.02.010

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, and
mediasiatica, which differ with regard to their biochemical
properties and geographical distribution (Keim et al., 2007;
Oyston, 2008). F. tularensis subsp. tularensis (type A) and
holarctica (type B) cause the majority of reported cases of
disease in humans, with subsp. tularensis causing the more
severe disease (Ellis et al., 2002). Molecular subtyping has
further separated F. tularensis subsp. tularensis into 2
genetically distinct clades, A.I and A.II, which display

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

differences in transmission, disease outcome, and geographic distribution (Farlow et al., 2005; Johansson et al., 2004;
Staples et al., 2006) F. tularensis strain SCHU S4 is the
proposed subsp. tularensis-type strain (Ellis et al., 2002).
F. tularensis is highly infectious, which has led many
nations including the USA, the former Soviet Union, and
Japan to regard it as a potential biological weapon (Dennis
et al., 2001). Diagnostic methods for the identification of
F. tularensis include culturing with cysteine-enriched
media and testing by fluorescent-labeled antibodies; however, these methods are either time consuming, not useful for
diagnosis of acute infections, or lacking in sensitivity
(Versage et al., 2003). The potential for F. tularensis as a
biological weapon has highlighted the need for rapid
diagnostics; hence, several real-time PCR assays have been
developed (Kugeler et al., 2006; Mitchell et al., 2010; Molins
et al., 2009; Tomaso et al., 2007; Versage et al., 2003). These
types of assays are used by select laboratories of the
Laboratory Response Network (LRN), a diagnostic laboratory network for potential bioterrorism threats, for the
presumptive identification of F. tularensis in clinical and
environmental specimens (Rotz & Hughes, 2004).
As the use of molecular diagnostics, such as rapid real-time
PCR assays, has become routine in diagnostic laboratories,
specimen throughput has increased accordingly (Schuurman
et al., 2007). Manual nucleic acid extraction methods have the
potential to be the rate-limiting component of rapid testing
protocols because of their limited throughput and labor
intensiveness (Knepp et al., 2003). Automated methods can
be a viable alternative to manual methods because they offer
medium- to high-throughput options for nucleic acid
extraction. It has been widely reported that the efficiency
of DNA extraction can influence the subsequent sensitivity
of PCR assays (Black & Foarde, 2007; Dauphin et al., 2009;
Durnez et al., 2009; Queipo-Ortuno et al., 2008; Schuurman
et al., 2005); therefore, the selection and use of optimal
extraction methods are crucial for laboratory diagnostics.
Few previous studies have evaluated DNA extraction
methods specifically for the isolation of F. tularensis DNA.
One study (Whitehouse & Hottel, 2007) compared commercial DNA extraction methods for the recovery of F. tularensis
DNA and reported that the UltraClean Microbial DNA
isolation kit (MoBio Laboratories, Carlsbad, CA) and the
PowerMax kit (MoBio Laboratories) were optimal among
those studied. This study (Whitehouse & Hottel, 2007) used
soil samples, which is an important sample type, but is only
one of the types submitted to laboratories that perform testing

for biothreat agents. Furthermore, the aforementioned study


compared manual DNA extraction kits, which do not provide
the high-throughput capacity that may be required in
laboratories that process large numbers of specimens.
The purpose of this study was to evaluate commercial
automated and manual extraction methods for the recovery
of F. tularensis DNA. Since environmental samples are
among the most common sample types submitted for testing
during suspected biothreat situations (Luna et al., 2003), and
culture is used for confirmatory testing, this study compared
6 methods, including 2 automated methods and 4 manual
methods, using bacterial suspensions and spiked swabs. The
evaluation included viability testing of the DNA extracts by
culture, limit of detection (LOD) studies for phosphatebuffered 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 performed
in a biosafety level 3 laboratory using relevant containment
and safety precautions, including the use of a powered airpurifying respirator.
2.2. F. tularensis strains and culture
The 7 F. tularensis strains used in this study (Table 1) were
provided by the Division of Vector-Borne Diseases, CDC
(Fort Collins, CO) and have been described in previous
reports (Molins-Schneekloth et al., 2008; Versage et al.,
2003). Six of the 7 strains were selected as representative of
the 2 F. tularensis subsp. which are clinically relevant, subsp.
tularensis (strains MA00-2987, SCHU S4, WY96-3418, and
NM99-1823) and subsp. holarctica (strains KY99-3387
and OR96-0246). The live-vaccine strain (LVS) was used
as a positive-control strain for real-time PCR assays.
F. tularensis cultures were initiated from lyophilized
stocks, which were reconstituted with 0.5 mL of Brain Heart
Infusion broth. The cultures were streaked for isolated
colonies onto cysteine heart agar supplemented with 9%
sheep blood (CHAB) plates, and the plates were incubated
for 48 h at 37 C. For each strain, a single colony was
transferred to 1 mL of sterile physiologic saline (0.85%
sodium chloride) using a sterile inoculating loop and mixed
by vortexing at low speed for 30 s. A 200-L aliquot of each

Table 1
Francisella tularensis strains used in this studya
Subspecies (type)

CDC accession numberb

Origin

F. tularensis subsp. tularensis (A.I)


F. tularensis subsp. tularensis (A.II)
F. tularensis subsp. holarctica (B)

MA00-2987, SCHU S4
WY96-3418, NM99-1823
KY99-3387, OR96-0246, LVS

Massachusetts, Ohio
Wyoming, New Mexico
Kentucky, Oregon, Russia

a
b

Strain information from Molins-Schneekloth et al. (2008) and Versage et al. (2003).
CDC accession number, excluding SCHU S4 and LVS, assigned by the Division of Vector-Borne Diseases, CDC, Fort Collins, CO.

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

suspension was then spread onto CHAB plates in triplicate


and the plates were incubated for 48 h at 37 C. Cultures
were harvested into 10 mL of sterile PBS (0.01 mmol/L, pH
7.4) using sterile Dacron fiber-tipped swabs (Fisher
Scientific, Pittsburgh, PA), which were pre-moistened with
PBS. To determine the bacterial concentration, serial 10-fold
dilutions of this suspension were performed in physiologic
saline, 100-L aliquots were spread onto CHAB plates in
triplicate, and the plates were incubated as described above.
Colonies were counted using standard microbiological
methods, and the quantified F. tularensis suspensions were
stored at 70 C until use.
2.3. Spiking of swabs
Three types of commonly used swabs were used for the
spiking experiments, cotton (Fisher Scientific), polyester
(Fisher), and foam (Epicentre Biotechnologies, Madison,
WI). Tenfold serial dilutions of F. tularensis strain SCHU
S4 with a starting concentration of 107 colony forming units
(CFU)/mL were performed in PBS, and the swabs were
inoculated in triplicate with 10-L aliquots at each
concentration. The swabs were allowed to air dry for 30
min at room temperature and were then processed for
F. tularensis recovery using the Swab Extraction Tube
System (SETS) (Roche Applied Sciences, Indianapolis, IN).
The SETS is a disposable centrifugal system consisting of an
inner tube (with a hole) and an outer collection tube, and its
use for the recovery of F. tularensis from swabs has been
described in detail previously (Walker et al., 2010). The
recovered F. tularensis PBS suspensions were immediately
used for the DNA extraction procedures.
2.4. Automated and manual DNA extraction methods
Six commercial DNA extraction methods, including 2
automated instruments and 4 manual kits, were evaluated in
this study. All of the procedures were performed in triplicate
using 200-L aliquots of F. tularensis as either quantified
PBS suspensions or samples recovered from spiked swabs.
Following the DNA extraction procedures, the PCR-ready
DNA samples were stored at 20 C until use.
The 2 automated DNA extraction methods used different
principles for DNA extraction. The MagNA Pure Compact
instrument (Roche) utilized magnetic bead technology,
whereas the QIAcube (Qiagen, Valencia, CA) employed a
silica spin-filter (column) technology. For the MagNA Pure
Compact instrument, DNA extractions were performed using
the MagNA Pure Compact Nucleic Acid Isolation Kit I
(Roche). As a safety precaution, an external lysis protocol
was performed by combining each sample with 300 L of
MagNA Pure LC DNA Isolation Kit I Lysis/Binding Buffer,
followed by incubation at room temperature for 30 min.
Immediately following the lysis procedure, the samples
underwent automated extraction on the MagNA Pure
Compact instrument using the DNA Blood External Lysis
Purification Protocol, with an elution volume of 100 L. For

301

the QIAcube instrument, DNA extractions were performed


using the QIAamp DNA Blood Mini Kit (Qiagen).
Automated DNA extraction on the QIAcube was performed
using the Blood and Body Fluid Spin Protocol with an
elution volume of 200 L.
The 4 manual DNA extraction kits used 3 different
principles for DNA extraction. Both the IT 1-2-3 DNA
Sample Purification Kit (Idaho Technology, Salt Lake City,
UT) and the UltraClean Microbial DNA Isolation Kit
(MoBio Laboratories) combine bead-beating and spincolumn technologies. The MasterPure Complete DNA and
RNA Purification Kit (Epicentre) uses a precipitation
methodology, whereas the QIAamp DNA Blood Mini Kit
(Qiagen) utilizes the same silica spin-filter technology as
noted above. All of the manual DNA extraction methods
were 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 inactivate
F. tularensis strains, the DNA extracts were tested for bacterial
viability. A total of 126 DNA samples were tested, which
included the following for each of the 6 DNA extraction
methods evaluated: triplicate sample extracts from F. tularensis strain SCHU S4 at a concentration of 106 CFU/mL (18
samples), and triplicate sample extracts from 6 of the
F. tularensis strains listed in Table 1 (MA00-2987, SCHU
S4, WY96-3418, NM99-1823, KY99-3387, and OR96-0246)
at a concentration of 105 CFU/mL (108 samples). Immediately
following each extraction procedure, 10% of the volume of
each sample extract was spread onto CHAB plates and the
plates were incubated for up to 5 days at 37 C. As a control for
this viability testing, an equal volume of each stock live
bacterial suspension was spread onto CHAB plates and the
plates were incubated as described above. Viability was
determined by observation of the plates for colonies. As an
additional safety precaution, the remaining volume of the
viability-tested extracts, as well as all other DNA extracts
prepared in this study, was micro-filtered using 0.1-m
centrifugal filter units (Millipore, Billerica, MA) using a
method described previously for removal of Bacillus anthracis
spores from DNA preparations (Dauphin & Bowen, 2009).
2.6. DNA yield and purity
DNA extracted from 3 F. tularensis strains, including 1
representative each for type A.I (SCHU S4), type A.II
(NM99-1823), and type B (KY99-3387), was quantified from
a concentration of bacteria at 106 CFU/mL using a NanoDrop
8000 Spectrophotometer (ND Technologies, Wilmington,
DE). The spectrophotometer was blanked before measurement with the corresponding elution buffer for each DNA
extraction method. For the MagNA Pure Compact, the
spectrophotometer was blanked with the elution buffer for the
MagNA Pure LC extraction kit. For each sample, the

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absorbance at 260 nm (A260) was measured. This value was


used to calculate the average concentration of DNA for each
set of triplicate samples by multiplying the A260 measurement
by a conversion factor (50 g/mL per 1 A260 unit for doublestranded DNA). To estimate the purity of DNA extracts, the
absorbance at 280 nm (A280) was measured also and the
average ratio between the A260 nm and A280 nm (A260/A280)
was calculated for triplicate samples. Samples with A260/A280
ratios between 1.8 and 2.0 were presumed to be free of
significant contamination (Manchester, 1995).
2.7. Preparation of positive controls
Cultures of F. tularensis strain LVS were prepared and
used as a positive control in real-time PCR assays. The
cultures were harvested into 250 L of sterile deionized water
in microcentrifuge tubes. The samples were vortexed briefly,
boiled for 5 min, and pelleted by centrifugation for 30 s at
10,000 g. The supernatants were transferred to 0.1-m filter
units and filtered as described previously. Filtered cell lysates
were diluted in Tris-EDTA buffer to dilutions which
produced real-time PCR cross-threshold (CT) values between
25 and 30 using the real-time PCR signature sets described
below. The positive-control samples were stored at 20 C
throughout this study.
2.8. Real-time PCR analysis
The multi-target real-time PCR assay described by
Versage et al. (2003) was used to evaluate the 6 DNA
extraction methods for the recovery of F. tularensis DNA
from PBS suspensions and spiked swabs. The assay was
developed for the specific detection of F. tularensis in
clinical and environmental specimens by targeting multiple
genetic loci: the ISFtu2 insertion element, the 23kDa gene,
and the tul4 gene (Versage et al., 2003). PCR was performed
using 25-L reaction volumes, each of which contained a
final concentration of 1X LightCycler FastStart DNA Master
HybProbes PCR Master Mix (Roche Molecular Biochemicals, Indianapolis, IN), 500 nmol/L of each PCR primer, 100
nmol/L of each FAM-labeled TaqMan probe, 4 mmol/L
MgCl2 (23kDa and tul4 assays) or 5 mmol/L MgCl2 (ISFtu2
assay), 0.5 U of uracil-DNA glycosylase (Roche), and 5 L
of either DNA extract, positive control DNA, or water (for
no-template controls). As an additional control, an internalpositive-control (IPC) real-time PCR assay (Applied Biosystems, Foster City, CA) was used to assess the ability of
each DNA extraction method to remove potential PCR
inhibitors. The IPC reagents included a control DNA, PCR
primers, and a VIC-labeled hydrolysis probe. These reagents
were added to each PCR reaction and were run in the
presence of each DNA extract.
Real-time PCR was performed on the 7500 Fast RealTime PCR System (Applied Biosystems) using the standard
7500 operational setting and a thermocycling profile
consisting of an incubation at 50 C for 2 min (glycosylase
step), a hot-start Taq activation step of 95 C for 10 min,

followed 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 were
performed using the 7500 Fast System Sequence Detection
Software, version 1.4, with the 21 CFR Part 11 electronic
records module.
To compare the automated and manual DNA extraction
methods for the recovery of DNA from F. tularensis, we
performed real-time PCR using triplicate DNA extracts
prepared from F. tularensis strain SCHU S4 at concentrations ranging from 106 to 100 CFU/mL. The LOD was
determined to be the lowest concentration for which 3 out of
3 replicates produced a positive result for all 3 real-time PCR
targets, as indicated by CT values of 40. To compare the
extraction methods for the recovery of DNA among
F. tularensis strains, real-time PCR was performed using
triplicate sample extracts prepared from 6 strains of
F. tularensis at a concentration of 105 CFU/mL. Comparisons for the isolation of F. tularensis DNA from swab
specimens were performed using triplicate DNA extracts
prepared from swabs spiked with dilutions of F. tularensis
strain SCHU S4. The LOD for spiked swabs was determined
as described above. All PCR runs were repeated 2 times for a
total of 3 PCR runs for each comparative evaluation.
2.9. Statistical analysis
To determine whether the variability of CT values for
F. tularensis DNA extracted from PBS suspensions and spiked
swab samples was significant, the mean CT values for each
method were compared using 1-way ANOVA (n = 27). This
analysis included data for triplicate sample extracts from strain
SCHU S4, which was tested in a 3-target real-time PCR assay
for a total of 3 PCR runs (9 data points per PCR run for each
extraction method). To determine whether the strain-to-strain
variability of CT values for DNA extracted from 6 F. tularensis
strains was significant, the CT values were compared using
1-way ANOVA (n = 54). This analysis included data for
triplicate sample extracts from the 6 strains, which were tested
in a single target real-time PCR assay for a total of 3 PCR
runs (18 data points per PCR run for each extraction method).
When significant differences were identified, Tukey's
multiple comparison test was used to perform nonparametric
pair-wise analyses of the CT values.

3. Results
3.1. Evaluation of DNA extraction methods for inactivation
of F. tularensis strains
All of the DNA extraction methods were highly efficient
at killing all F. tularensis strains at concentrations up to 106
CFU/mL as there was no growth observed in cultures of such
DNA extracts prepared using each of the 6 methods. The
viability testing controls were positive for each strain of
F. tularensis tested. Since 10% of the volume of each DNA
extract was used for viability testing, this would indicate at

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

Extraction
methoda
MagNA Pure
Compact
QIAcube
IT 1-2-3
MasterPure
QIAamp
UltraClean

LOD
(CFU/mL)b

Average CT (mean SD)c,d


ISFtu2

23kDa

tul4

10

29.6 1.35

32.8 0.73

33.0 0.30

103
105
103
103
104

31.0 0.27
32.5 1.42
29.3 0.29
30.8 0.60
29.0 1.16

34.8 0.65
32.3 1.64
31.8 0.96
33.6 1.12
32.9 1.15

34.5 0.65
34.9 0.85
32.0 0.72
36.1 3.65
33.0 2.14

a
Extraction methods were performed in triplicate using F. tularensis
strain SCHU S4 at concentrations ranging from 106 to 100 CFU/mL.
b
The LOD was determined to be the lowest concentration for which 3
out of 3 replicates produced a positive result for the 3 real-time PCR targets
as described by Versage et al. (2003).
c
The mean CT values (at the determined LOD) are shown to
demonstrate levels of reproducibility among replicate sample DNA extracts.
d
The differences in mean CT values between the 6 methods were found
to be significant by 1-way ANOVA (P = 0.001; n = 27). Pair-wise
comparisons using Tukey's multiple comparison test revealed significant
differences between the IT 1-2-3 kit and the UltraClean kit and each of the
other 4 extraction methods (P b 0.05; n = 27).

least a 5-log reduction of viability for the 6 DNA extraction


methods 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 DNA
extracted from F. tularensis strain SCHU S4 at concentrations ranging from 106 to 100 CFU/mL with the 6 DNA
extraction methods. DNA extraction using the MagNA Pure
Compact, the QIAcube, the MasterPure kit, and the QIAamp
kit yielded the most sensitive levels of detection by real-time
PCR, followed by the UltraClean kit, with the IT 1-2-3 kit
yielding DNA with the poorest level of detection. The LOD
was 103 (CFU/mL in the original cell suspension) for the
DNA extracted with the 4 methods which performed the
best. Since a 5-L volume of DNA extract was used for PCR
reactions, this would translate to 5 CFU per PCR reaction for
these 4 methods. The UltraClean kit yielded DNA which
resulted in a LOD of 104 CFU/mL (50 CFU/reaction), and
the PCR LOD for DNA prepared with the IT 1-2-3 kit was
105 CFU/mL (500 CFU/reaction). The differences in mean
CT values for the 6 DNA extraction methods were found to
be significant by 1-way ANOVA (P = 0.001; n = 27). Pairwise comparisons of CT values indicated significant
differences between the UltraClean kit and the IT 1-2-3
kit, 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 6
strains of F. tularensis
Using the real-time PCR results obtained with the index
strain, SCHU S4, as the baseline LOD, we evaluated the 6

DNA extraction methods for their efficiency of DNA


extraction from 6 different strains of F. tularensis. Fig. 1
shows the range of CT values for the ISFtu2 gene target from
the 6 strains at a concentration of 105 CFU/mL. The
MasterPure kit yielded DNA having the lowest average CT
values (equating to the best quality/quantity), with a triplicate
sample range (mean) of 19.725.9 (22.9), followed by the
QIAcube and the MagNA Pure Compact, with CT value
ranges (mean) of 20.826.5 (23.6) and 20.726.4 (23.9),
respectively. DNA extraction using the QIAamp kit and the
UltraClean kit resulted in CT values with ranges (mean) of
20.927.2 (24.1) and 24.031.6 (27.2), respectively. The IT 12-3 kit yielded DNA with the highest CT values (28.437.3)
and a mean CT value of 32.5. The differences in mean CT
values for the 6 DNA extraction methods were found to be
significant by 1-way ANOVA (P = 0.0001; n = 54). Pair-wise
comparisons of CT values indicated significant differences
between the UltraClean kit and the IT 1-2-3 kit when compared
to the 4 other DNA extraction methods (P b 0.05; n = 54).
3.4. Comparison of extraction methods for DNA yield
and purity
Table 3 shows the DNA extract volumes, average DNA
concentrations, yields, and the range of A260/A280 ratios for
the 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 to
200 L, with the MasterPure kit producing the smallest DNA
volume and the QIAcube and the QIAamp kit providing the
40

Average CT

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

303

30

20

10
MPC

QC

IT

MP

UC

Extraction Method
Fig. 1. Box-and-whisker plots showing the distribution of CT values
obtained using DNA prepared from 6 strains of F. tularensis at
concentrations of 105 CFU/mL using 2 automated DNA extraction methods,
the MagNA Pure Compact (MPC) and the QIAcube (QC), and 4 manual
DNA extraction methods, the IT 1-2-3 kit (IT), the MasterPure kit (MP), the
QIAamp kit (Q), and the UltraClean kit (UC). The middle line for each box
corresponds to the mean for the observed values. The end points of the
whiskers show the 2.5 and 97.5 percentiles. The differences in mean CT
values between the 6 DNA extraction methods were found to be significant
by 1-way ANOVA (P b 0.0001; n = 54). Tukey's multiple comparison test
revealed significant differences between the mean CT values for the
UltraClean kit and the IT 1-2-3 kit, when compared to the 4 other DNA
extraction kits (P b 0.05; n = 54).

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

Table 3
Comparison of sample volume, DNA concentration, yield, and purity
between automated and manual DNA extraction methods
Extraction
method

Volume
(L)

A260/A280
Concentration Total
(ng/L)
yield (g)a ratiosb

MagNA
Pure Compact
QIAcube
IT 1-2-3
MasterPure
QIAamp
UltraClean

100

16.10

1.61

1.561.79

200
200
50c
200
50

10.68
6.94
23.22
11.14
19.64

2.14
1.39
1.16
2.23
0.98

1.621.89
1.261.48
1.691.75
1.651.99
1.701.88

a
Total DNA yields were calculated from mean A260 measurements for
triplicate sample extracts from F. tularensis strains SCHU S4, NM99-1823,
and KY99-3387 at an input concentration of 106 CFU/mL, multiplied by the
conversion 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 triplicate
sample extracts from F. tularensis strain SCHU S4 at concentrations ranging
from 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 sufficient
for statistical analyses.

greatest volumes. The MasterPure kit yielded DNA with the


highest DNA concentrations for all F. tularensis strains. The
UltraClean kit yielded DNA at the second-highest concentration. The MagNA Pure Compact ranked third, followed
by the QIAamp kit and the QIAcube instrument. The IT 1-23 kit yielded the lowest concentration of DNA. The
automated QIAcube and the manual QIAamp kit resulted
in the greatest total DNA yields , followed by the MagNA
Pure Compact. The UltraClean kit resulted in the lowest total
DNA yield. The MasterPure, QIAamp, and UltraClean kits
produced 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
to 1.88, respectively.
3.5. Real-time PCR analysis of F. tularensis DNA recovered
from spiked swabs
Table 4 shows the real-time PCR LOD for DNA extracted
from spiked swabs. The 6 extraction methods resulted in
varying levels of detection among the 3 swab materials. For
cotton swabs, the MagNA Pure Compact and the QIAamp
kit performed best, with a LOD of 104 CFU/mL (100 CFU/
swab). The QIAcube, the MasterPure kit, and the UltraClean
kit 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 level
of detection (107 CFU/mL, 105 CFU/swab). For foam swabs,
all of the DNA extraction methods resulted in a LOD of
105 CFU/mL (103 CFU/swab), with the exception of the IT
1-2-3 kit, which resulted in the poorest level of detection
(106 CFU/mL, 104 CFU/swab). Likewise, for polyester
swabs, all of the DNA extraction methods resulted in a LOD
of 105 CFU/mL (103 CFU/swab), except the IT-1-2-3 kit,
which produced a LOD of 107 CFU/mL (105 CFU/swab).

There was no evidence of PCR inhibition for any of the DNA


extraction methods in samples recovered from the 3 swab
materials, as measured by the IPC assay (data not shown).

4. Discussion
The increasing availability of commercial DNA extraction methods emphasizes the need for a timely evaluation of
representative DNA extraction technologies with a range of
sample throughput capacities. This study demonstrated that
the sensitivity of F. tularensis detection by real-time PCR
was equivalent after DNA extraction by both automated
methods tested and by the MasterPure and the QIAamp
manual methods. Several studies have reported improved
PCR sensitivity using DNA extraction kits such as the
MasterPure kit, the QIAamp kit, and the UltraClean kit
(Dauphin et al., 2010; Knepp et al., 2003; Queipo-Ortuno
et al., 2008). The results of F. tularensis real-time PCR were
comparable for the MagNA Pure Compact and the QIAcube
instruments and the MasterPure and QIAamp kits because all
of these methods effectively extracted F. tularensis at
concentrations as low as 103 CFU/mL. Furthermore, the 2
automated methods resulted in significantly better real-time
PCR detection levels when compared to the IT 1-2-3 kit and
the UltraClean kit (Table 2). These results indicate that either
of the 2 automated DNA extraction methods is a suitable
alternative to the manual DNA extraction methods tested in
this study.
Several factors, such as DNA purity, concentration, and
damage, can influence the sensitivity of real-time PCR
assays. The results of this study indicated that DNA purity
had a likely influence on the sensitivity of the real-time PCR
assay. All of the methods resulted in comparable A260/A280
ratios, with the exception of the IT 1-2-3 kit, which had the
poorest values in comparison, and yielded DNA which was
subsequently detected less efficiently. There was also, as

Table 4
Real-time PCR LOD for DNA extracted from spiked swabs using automated
and manual DNA extraction methods
Extraction methoda

LODb,c
Cotton swabs

MagNA Pure Compact


QIAcube
IT 1-2-3
MasterPure
QIAamp
UltraClean

10 (100)
105 (103)
107 (105)
105 (103)
104 (100)
105 (103)

Foam swabs
5

10 (10 )
105 (103)
106 (104)
105 (103)
105 (103)
105 (103)

Polyester swabs
105 (103)
105 (103)
107 (105)
105 (103)
105 (103)
105 (103)

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

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

expected, an apparent correlation between DNA concentration and PCR sensitivity. With the exception of the
UltraClean kit, the DNA extraction methods which resulted
in the greatest concentrations also resulted in the best
levels of detection by real-time PCR. With unique regard
to the UltraClean kit, neither DNA purity nor DNA concentration appeared to correlate with PCR sensitivity for
F. tularensis suspensions, as this kit yielded better A260/A280
ratios and greater DNA concentrations than most of the
methods evaluated in this study, yet the DNA it yielded was
detected among the worst in comparison (Table 2). One
factor which may have contributed to the lower PCR
sensitivity observed with the UltraClean kit is DNA damage.
The lysis procedure for the UltraClean kit employed a
10-min bead-beating procedure. Mechanical disruption of
F. tularensis cells during the extraction process may have
resulted in DNA shearing and damage to PCR target regions
which can adversely affect PCR amplification (Scupham et
al., 2007). Furthermore, the instruction manual for this kit
offers an alternative lysis method for less DNA shearing,
indicating that the manufacturers are cognizant that the
bead-beading procedure may not be as appropriate for
some applications.
The ability of extraction methods to purify nucleic acid
and efficiently remove inhibitors from various sample
matrices is essential when using molecular diagnostics
such as PCR assays (Boom et al., 1999; Chan et al., 2008).
In this study, the automated and manual DNA extraction
methods were compared using spiked swab samples to
simulate specimens which may be tested in diagnostic
laboratories that perform testing for F. tularensis. The results
indicated that the 2 automated methods performed as well as
or better than the manual extraction methods for the recovery
of quality DNA from swab samples (Table 4). Moreover, the
results suggest that the automated methods were as efficient
as the manual methods for yielding DNA free of inhibitory
substances, as indicated by the IPC assay. Finally, the
efficiency of DNA extraction for the 2 automated methods
was not compromised when the methods were evaluated for
extraction from different swab materials. Combined, these
data indicate that the MagNA Pure Compact and the
QIAcube methods are as sufficient as the manual DNA
extraction methods used in this study for the isolation of
F. tularensis DNA from swabs.
One limitation of this study is that spiked swabs were
used to simulate environmental samples that would be
processed during laboratory bioterrorism-related investigations. It is not clear whether the results would be equivalent
for field-collected environmental samples. In addition, the
swabs were processed for testing shortly after inoculation.
Future studies should assess equivalence using environmental swabs obtained from surface sampling and assess the
impact of storage times on pathogen detection levels.
The aim of this study was to compare 2 automated DNA
extraction methods to manual methods for extraction of
F. tularensis DNA. The results show that the MagNA Pure

305

Compact and the QIAcube instruments perform equivalently


to each other and to other popular commercially available
manual methods. These 2 instruments offer diagnostic
laboratories the option to select automated methods for the
extraction of F. tularensis DNA with optimal PCR sensitivity.
Acknowledgments
The authors acknowledge Liberty Bost, Rebecca Hutchins, and Michael Vick for laboratory assistance. The authors
also thank Gregory Buzard, Pamela Diaz, and Harvey
Holmes for editorial comments.
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