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