JOURNAL OF CLINICAL MICROBIOLOGY, May 2005, p. 2471–2473 Vol. 43, No.

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0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.5.2471–2473.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Comparison of Six Methods of Extracting Mycobacterium tuberculosis

DNA from Processed Sputum for Testing by Quantitative
Real-Time PCR
Wade K. Aldous, June I. Pounder,* Joann L. Cloud, and Gail L. Woods
ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way,
Salt Lake City, Utah 84108
Received 18 October 2004/Returned for modification 10 January 2005/Accepted 18 January 2005

Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were

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compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)–10% Triton X with and
without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all
included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis
ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart
Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding
Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect
amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments.
Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4,
28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run
on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction
methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can
be completed in less than 1 h versus 2.5ⴚ3 h for the QIAGEN extraction.

Tuberculosis is a public health problem worldwide, and for
optimal control, early diagnosis is necessary (4, 6, 7). Several
researchers have developed real-time PCR assays that provide
rapid detection of various target sequences of Mycobacterium
tuberculosis complex (MTBC) and drug resistance genes in
patient specimens (1, 3, 5, 16–19). The ability of these assays to
detect MTBC in clinical samples is dependent on both the
target sequence selected and the efficiency of the DNA extrac-
tion procedure. Several methods of mycobacterial cell wall lysis
and DNA extraction have been evaluated, including deter-
gents, proteolytic enzymes, mechanical disruption, and tem-
perature changes alone and in various combinations (1, 2, 5,
8–15, 17, 20). The objective of this study was to compare six
methods of extracting M. tuberculosis DNA from respiratory
specimens: Tris-EDTA (TE) boil extraction (10), PrepMan
ultra extraction (Applied Biosystems, Inc., Foster City, CA),
Infectio Diagnostics, Inc. (IDI) lysis extraction (Infectio Diag-
nostics, Inc. Quebec, Canada), QIAGEN QIAmp DNA mini
kit (QIAGEN, Inc., Valencia, CA), sodium dodecyl sulfate
(SDS)–Triton X extraction (9), and SDS–Triton X plus soni-
cation. The effectiveness of each extraction method was as-
sessed using two quantitative real-time PCR assays.
Sample preparation. Digested and decontaminated (N-
acetyl-cysteine–2% NaOH) sputum specimens that were cul-
ture negative for mycobacteria were pooled for use as the
standard respiratory specimen. A suspension of M. tuberculosis
ATCC 27294 was prepared in sterile saline and adjusted to the
density of a 1.0 McFarland standard. The suspension was di-
* Corresponding author. Mailing address: ARUP Institute for Clin-
ical and Experimental Pathology, 500 Chipeta Way, Salt Lake City,
Utah 84108. Phone: (801) 583-2787, ext. 3223. Fax: (801) 584-5109.
E-mail: june.pounder@aruplab.com.
luted 1:10 in saline and used to spike the pooled respiratory
specimen. Spiked specimens were stored in 200-␮l aliquots at
⫺70°C until extracted.
DNA extraction. For all six extraction procedures, each of
which was repeated eight times, the spiked respiratory speci-
men was first thawed and centrifuged at 6,000 ⫻ g for 1 min.
The supernatant was discarded, and the pellet was processed
for each procedure as follows. (i) TE boil extraction. A 200-␮l
aliquot of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA)
was added, and the mixture was briefly mixed on a vortex
mixer. The suspension was placed in a boiling water bath for 15
min to destroy any viable mycobacteria and then centrifuged at
16,000 ⫻ g for 5 min. A 100-␮l aliquot of the supernatant was
transferred to a sterile tube and stored at ⫺20°C until PCR
testing. (ii) PrepMan extraction. A 200-␮l aliquot of PrepMan
Ultra sample preparation reagent were added to the pellet,
and the suspension was treated as described for TE buffer. (iii)
IDI extraction. Pellets were treated as described for TE buffer
with the following additional steps. The TE suspension was
placed into an IDI lysis tube (Infectio Diagnostic, Inc., Que-
bec, Canada), which contains a glass bead matrix. Tubes were
vigorously mixed for 5 min on the highest setting of a Vortex
Genie 2 (Scientific Industries, Inc., Bohemia, NY) in a micro-
tube foam insert and then placed into a boiling water bath. (iv)
QIAGEN extraction. Pellets were processed using the
QIAGEN QIAamp DNA mini kit (QIAGEN, Valencia, CA)
tissue protocol, with the following modifications: an enzymatic
digestion step with 30 mg/ml lysozyme added to the tissue lysis
buffer was followed by boiling for 15 min, and the proteinase K
step was incubated at 56°C for 1 h. DNA was purified per
manufacturer’s recommendations through a spin column. (v)
SDS–Triton X extraction. Pellets were treated as described for
TE buffer, except 200 ␮l of the nonionic detergent mix (2%

2471
2472 NOTES
SDS–10% Triton X-100) was substituted for the TE buffer (9).
(vi) SDS–Triton X plus sonication extraction. A 200-␮l aliquot
of 2% SDS–10% Triton X-100 was added to the pellet, and the
mixture was briefly agitated on a vortex mixer. The suspension
was sonicated for 15 min and then treated as described for TE
buffer.
Primers and probes. Primers and probes were designed us-
ing minor grove binding (MGB) Eclipse Design Software (Ep-
och Biosciences, Bothell, WA). Primers were purchased from
IT BioChem (Idaho Technologies, Salt Lake City, UT), and
the minor grove binding (MGB) Eclipse probes were from
Epoch Biosciences. MTBC-specific primers were used to am-
plify an 83-bp fragment of the IS6110 target. The forward
primer corresponds to the region from base 801 to base 818
(sequence, 5⬘-CTAACCGGCTGTGGGTA-3⬘; base number-
ing referenced with GenBank accession no. X52471). The re-
verse primer corresponds to the region of IS6110 from base
866 to base 884 (sequence, 5⬘-CGTAGGCGTCGGTGACAA
A-3⬘). The MTBC-specific Eclipse probe consists of an oligo-
nucleotide (sequence, 5⬘-MGB-NFQ-AGACCTCA*CCTAT*
GT-FAM-3⬘) labeled with a nonfluorescent quencher molecule
and the MGB moiety at the 5⬘ end and a FAM fluorescent dye
label at the 3⬘ end. Bases with asterisks (*) are proprietary
“superbases,” manufactured by Epoch Biosciences for in-
creased hybridization stability.
An internal control (IC) plasmid containing genomic DNA
from nonhuman sources was used to detect PCR inhibition in
the extracts. The IC primers and Eclipse probe have been
described previously (J. B. Stevenson, K. C. Carroll, and D. R.
Hillyard, Abstr. 18th Ann. Clin. Virol. Symp., abstr. M35,
2002).
Real-time PCR conditions. Each extract was tested on two
real-time PCR instruments: the Smart Cycler II System (Ce-
pheid, Sunnyvale, CA) and the Rotor-Gene 3000 real-time
DNA analysis system (Corbett Research, Sydney, Australia).
All reactions on each instrument were optimized to obtain the
best amplification kinetics. For the Smart Cycler and the Ro-
tor-Gene, the PCR mixture (25 ␮l) contained 12.5 ␮l of re-
constituted OmniMix HS (3 U TaKaRa hot start Taq polymer-
ase, 200 ␮M deoxynucleoside triphosphates, 4 mM MgCl2, and
25 mM HEPES buffer [pH 8.0 ⫾ 0.1]), 0.5 ␮M of each MTBC
primer, 200 nM MTBC Eclipse probe, 0.25 ␮M of each IC
primer, 200 nM IC Eclipse probe, 20 pg IC DNA, and 2 ␮l
extracted DNA template with the following exception. The
Rotor-Gene PCR mixture contained 300 nM MTBC Eclipse
probe. Water was used as a no-template control. Cycling con-
ditions were identical for both instruments: 1 cycle at 95°C for
2 min, 50 cycles at 95°C for 5 s, 58°C for 20 s, and 76°C for 20 s.
The crossing threshold cycle (Ct) is the cycle at which there is
a significant increase in fluorescence above the background or
a specified threshold. The Ct was determined from a curve
generated from a plot of cycle number versus fluorescence with
a manual threshold set above the background fluorescence of
the no template control. The Ct is inversely proportional to the
logarithm of the initial number of template molecules.
DNA standards. M. tuberculosis ATCC 27294 DNA was ex-
tracted using the IDI procedure. The concentrations of M.
tuberculosis and IC DNA were determined by using the Pi-
coGreen dsDNAQuantitation kit (Molecular Probes, Inc., Eu-
gene, OR). Serial 10-fold dilutions of the M. tuberculosis DNA,
J. CLIN. MICROBIOL.
FIG. 1. Mean Ct (⫾SD) for four extraction methods on the Smart
Cycler and Rotor-Gene 3000.
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ranging from 25 pg/␮l to 2.5 fg/␮l, were used as external stan-
dards. These standards were run in triplicate on both instru-
ments. A standard curve was included or imported for each
Rotor-Gene run. To generate the standard curve, the Ct value
in the logarithmic linear phase was plotted against the loga-
rithm of the known concentrations of M. tuberculosis DNA.
The concentrations of mycobacterial DNA in the spiked respi-
ratory samples were calculated by comparing the average Ct
value from the logarithmic linear phase of the fluorescence
curve with the line generated from external standards on that
instrument.
Statistical analysis. The efficiency of each DNA extraction
method (n ⫽ 8) was compared using the data analysis package
included within Microsoft Excel 2000 software (Microsoft Cor-
poration, Redmond, WA). All methods on both instruments
were evaluated by a two-tailed Student’s t test paired two-
sample means.
The limit of detection of both instruments, using known
concentrations of M. tuberculosis DNA to create a standard
curve, was 0.5 fg of DNA in the presence of an IC. The PCR
assay was inhibited on both instruments when specimens were
extracted using 2% SDS–10% Triton X with and without son-
ication, as indicated by the lack of amplification of the IC
target in the extracted samples and amplification of IC in the
no-template control samples. These methods were included in
our evaluation based on data from a report by Khan and Yadav
(9), which showed that the SDS and Triton X concentrations
we used were optimal for extracting DNA from a mycobacte-
rial colony. In retrospect, our results are not totally unex-
pected, since an excess of SDS above 0.01% has been shown to
inhibit PCR, primarily due to denaturation of the Taq poly-
merase (Critical Factors for Successful PCR manual,
QIAGEN, Inc.). Although there were methodological differ-
ences between our study and that of Khan and Yadav (e.g.,
standard versus real-time PCR, different polymerases, different
ratios of template to reaction volume), they do not adequately
explain the complete inhibition observed in our evaluation and
the amplification in theirs. Reasons for the striking difference
between the results of the two studies are unclear.
Mean (⫾ standard deviation [SD]) Ct values for the other
four extraction methods evaluated (illustrated in Fig. 1) were
25.3 ⫾ 0.64 (range, 24.2 to 26.6) for IDI, 25.9 ⫾ 0.62 (range,
24.8 to 26.7) for QIAGEN, 25.8 ⫾ 0.56 (range, 24.7 to 26.6) for
PrepMan, and 27.9 ⫾ 0.79 (range, 26.7 to 29.6) for TE when
VOL. 43, 2005
testing was performed on the Smart Cycler. When testing was
done on the Rotor-Gene, mean (⫾SD) Ct values were 25.1 ⫾
1.82 (range, 21.7 to 27.5) for IDI, 26.9 ⫾ 1.61 (range, 23.9 to
29.4) for QIAGEN, 26.5 ⫾ 1.52 (range, 26.3 to 29.7) for Prep-
Man, and 27.7 ⫾ 1.10 (range, 23.7 to 29.8) for TE. The differ-
ences in Ct values were significant (P ⫽ 0.05) for IDI and TE,
QIAGEN and TE, and PrepMan and TE when testing was
performed on the Smart Cycler, but there were no significant
differences in Ct values between any of the methods when
testing on the Rotor-Gene.
The amount of DNA extracted by each method, which is
inversely proportional to Ct, was calculated from the mean Ct.
When testing on the Smart Cycler, the DNA yield was 42.8 pg
for IDI, 28.2 pg for QIAGEN, 30.4 pg for PrepMan, and 7.4 pg
for TE. DNA yields for these same methods when testing on
the Rotor-Gene were 51.7 pg, 14.9 pg, 20.1 pg, and 8.6 pg,
respectively.
Column-purified DNA theoretically should be the cleanest,
containing the fewest PCR-inhibitory substances. The goal of
our study was to find a DNA extraction method that was quick
but did not compromise the PCR or assay sensitivity, using the
QIAGEN extraction method as the gold standard for PCR
purification. Our results showed that column purification is not
necessary for the extraction of DNA from sputum samples to
be tested for M. tuberculosis by PCR. The time required to
complete each extraction was less than 1 h for TE buffer,
PrepMan, and IDI and involved two or three transfers of
material to new tubes, compared to 2.5 to 3 h for QIAGEN
with extensive hands-on processing.
Although not the primary aim of our study, we evaluated two
different real-time PCR instruments for the detection of M.
tuberculosis from respiratory samples in a clinical laboratory
setting. PCR on the Smart Cycler was completed in 55 min,
and the Rotor-Gene PCR runs were 95 min in duration. The
Smart Cycler can run 16 reactions wells per block with up to six
processing blocks connected to one computer for a 96-tube
capacity. The Rotor-Gene has a 72 tube capacity for a 25-␮l
PCR volume. The Smart Cycler is a totally closed system with
a lower chance of amplicon tubes opening for possible ampli-
con contamination.
A final point to be considered when selecting an extraction
method for use with the M. tuberculosis PCR assay is cost. For
the kits or reagents evaluated in this study, the cost per extrac-
tion is $3.00 for IDI, $2.30 for QIAGEN, $1.05 for PrepMan,
and approximately $0.03 for TE buffer.
In summary, of the six extraction methods evaluated, IDI
lysis tubes provided the greatest yield of DNA on both the
Smart Cycler and Rotor-Gene instruments. Additionally, the
IDI procedure was technically simple and was completed in
less than 1 h, with approximately 20 min of hands-on time.
Extraction with TE buffer, although very easy to do and inex-
pensive, provided the poorest DNA yield. SDS–Triton X con-
sistently inhibited the PCR on both real-time instruments and,
therefore, cannot be recommended for extraction of mycobac-
terial DNA.
We thank Andrea Stetler for secretarial assistance, Mark Kushnir
for the statistical advice, and Jeffery Stevenson for providing the in-
ternal control plasmid. Yevgeniy S. Belousov at Epoch Bioscience
designed the MGB primers and probe used in this study.
NOTES 2473
This study was funded by the Associated Regional and University
Pathologists Institute for Clinical and Experimental Pathology.
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