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Journal of Clinical Microbiology, November 2001, p. 4131-4137, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4131-4137.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Rifampin Resistance in
Mycobacterium tuberculosis in a Single Tube with
Molecular Beacons
Hiyam H.
El-Hajj,1
Salvatore A. E.
Marras,2
Sanjay
Tyagi,2
Fred Russell
Kramer,2,* and
David
Alland1
Department of Medicine, Montefiore Medical
Center, Bronx,1 and Department of
Molecular Genetics, Public Health Research Institute, New
York,2 New York
Received 1 June 2001/Returned for modification 13 August
2001/Accepted 26 August 2001
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ABSTRACT |
Current clinical assays for determining antibiotic susceptibility
in Mycobacterium tuberculosis require many weeks to
complete due to the slow growth of the bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h
and reliably identifies rifampin-resistant M.
tuberculosis in DNA extracted directly from sputum. Ninety-five
percent of mutations associated with rifampin resistance occur in an
81-bp core region of the bacterial RNA polymerase gene,
rpoB. All mutations that occur within this region result
in rifampin resistance. The assay uses novel nucleic acid hybridization
probes called molecular beacons. Five different probes are used in the
same reaction, each perfectly complementary to a different target
sequence within the rpoB gene of rifampin-susceptible
bacilli and each labeled with a differently colored fluorophore.
Together, their target sequences encompass the entire core region. The
generation of all five fluorescent colors during PCR amplification
indicates that rifampin-susceptible M. tuberculosis is
present. The presence of any mutation in the core region prevents the
binding of one of the molecular beacons, resulting in the absence of
one of the five fluorescent colors. When 148 M.
tuberculosis clinical isolates of known susceptibility to
rifampin were tested, mutations associated with rifampin resistance
were detected in 63 of the 65 rifampin-resistant isolates, and no
mutations were found in any of the 83 rifampin-susceptible isolates.
When DNA extracted directly from the sputum of 11 patients infected
with rifampin-resistant tuberculosis was tested, mutations were
detected in all of the samples. The use of this rapid assay should
enable early detection and treatment of drug-resistant tuberculosis in
clinical settings.
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INTRODUCTION |
The worldwide increase in drug-resistant
tuberculosis poses a major public health threat (16).
First-line antituberculosis treatment often fails in patients with
rifampin-monoresistant or multidrug-resistant tuberculosis
(13). Inappropriate treatment can result in the
development of resistance to additional antibiotics (3, 4)
and in increased mortality (15, 26). Because Mycobacterium tuberculosis grows extremely slowly (5,
17), conventional susceptibility testing can require many weeks
to complete (9). There is thus an urgent need to develop a
rapid, simple, and accurate assay to assess drug resistance in M. tuberculosis (7).
Rifampin resistance is an excellent marker for multidrug-resistant
tuberculosis because all of these strains are resistant to rifampin
(23). Therefore, a screening assay does not need to test
susceptibility to all antituberculosis drugs. Rifampin resistance is
particularly amenable to detection by rapid genotypic assays because
95% of all rifampin-resistant strains contain mutations localized in
an 81-bp region of the bacterial RNA polymerase gene, rpoB,
which encodes the active site of the enzyme (14, 21). Moreover, all mutations that occur in this region result in rifampin resistance. By contrast, all rifampin-susceptible M. tuberculosis isolates have the same wild-type nucleotide
sequence in this region (14, 22). Thus, it is only
necessary to detect a mutation in the rpoB core region to
know that the bacilli are rifampin resistant. A number of novel assays
based on the PCR have been developed to detect these mutations
(2, 24, 25). However, they are technically difficult and
have been of limited clinical utility.
We have now developed a single-tube PCR assay that detects all
mutations that occur in the M. tuberculosis rpoB core region in real time. The assay is simple, rapid, specific, and extremely sensitive. In less than 3 h, it reliably detects resistance
mutations in DNA extracted directly from sputum. The assay is based on
previous work of members of our group that demonstrated that
fluorogenic nucleic acid hybridization probes, called molecular beacons
(27), can be used to detect mutations in the M. tuberculosis rpoB gene (18, 19). The assay utilizes
five differently colored molecular beacons, each of which binds to a
different target segment within the rpoB core region.
Together, the five probes interrogate the entire 81-bp core. Each
molecular beacon was designed to be so specific that it does not bind
to its target if the target sequence differs from the
rifampin-susceptible sequence by as little as a single nucleotide
substitution (12, 28). Since molecular beacons fluoresce
only when they are bound to their targets, the absence of any one of
the five colors in the assay indicates that the bacilli in the sample
are rifampin resistant.
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MATERIALS AND METHODS |
Molecular beacons and primers.
Both conventional and
wavelength-shifting molecular beacons were prepared by solid-phase DNA
synthesis on an Applied Biosystems (Foster City, Calif.) 394 DNA/RNA
synthesizer. The nucleotide sequences of the six molecular beacons that
were synthesized were as follows: Probe A, 5'-Texas
red-TTTTTT-fluorescein-CGAGCTCAGCTGGCTGGTGCGCTCG-dabcyl-3'; Probe B,
5'-tetrachlorofluorescein-GCTACGGAGCCAATTCATGGACCAGACGTAGC-dabcyl-3'; Probe C,
5'-tetramethylrhodamineTTTTTT-fluorescein-CCGACGCCGACAGCGGGTTGTTCGTCGG-dabcyl-3'; Probe D,
5'-rhodamine-TTTTTT-fluorescein-CCACGCTTGTGGGTCAACCCCCGTGG-dabcyl-3'; Probe E,
5'-fluorescein-CCTGCCGCCGACTGTCGGCGCTGGCAGG-dabcyl-3'; and a 16S probe,
5'-fluorescein-GCGCCCGCGGCCTATCAGCTTGTTGGTGGCGC-dabcyl-3'; underlines identify the arm sequences.
During synthesis, controlled-pore glass columns (Biosearch
Technologies, Novato, Calif.) were used to incorporate dabcyl at the 3'
end of the oligodeoxyribonucleotides, fluorescein phosphoramidites (Glen Research, Sterling, Va.) were used to incorporate internal fluorescein moieties, and tetrachlorofluorescein phosphoramidites were
used to incorporate tetrachlorofluorescein at the 5' end. A
thiolmodifier phosphoramidite (Glen Research) was incorporated at the
5'-terminal position when fluorescein or tetramethylrhodamine was used
as a label, while an aminomodifier phosphoramidite (Glen Research) was
incorporated at the 5'-terminal position when rhodamine or Texas red
was used as a label. Iodoacetylated fluorescein or tetramethylrhodamine
(Molecular Probes, Eugene, Ore.) was then coupled to the 5'-thiol
groups, or succinimidyl esters of rhodamine or Texas red (Molecular
Probes) were coupled to the 5'-amino groups. All probes were purified
by gel exclusion chromatography through a NAP-5 Sephadex column
(Amersham Pharmacia Biotech, Piscataway, N.J.), followed by
high-pressure liquid chromatography on a Beckman Coulter (Fullerton,
Calif.) System Gold chromatograph through a C-18 reverse-phase column
(Waters Corporation, Milford, Mass.). A detailed protocol for molecular
beacon synthesis is available (www.molecular-beacons.org).
The nucleotide sequences of the primers that were used to amplify a
189-bp segment of the
rpoB gene that contains the entire
core region were 5'-GGAGGCGATCACACCGCAGACGTT-3' and
5'-ACCTCCAGCCCGGCACGCTCACGT-3';
and the nucleotide sequences
of the primers that were used to
amplify a 209-bp segment of the
mycobacterial 16S rRNA gene that
contains a conserved sequence were
5'-GAGATACTCGAGTGGCGAAC-3'
and
5'-GGCCGGCTACCCGTCGTC-3'.
Sample preparation.
One or two colonies of previously
well-characterized M. tuberculosis clinical isolates that
were grown on Löwenstein-Jensen slants were lysed by boiling in 1 ml of H2O for 20 min. Five microliters of each
lysate was used as a template for each PCR assay.
Purified DNA from other
Mycobacterium species was also used
in the assay. The species were the following:
M. africanum,
M. asiaticum,
M. avium,
M. celatum,
M. chelonae,
M. flavescens,
M. fortuitum,
M. gastri,
M. genavense,
M. intracellulare,
M. kansasii,
M. leprae,
M. lufu,
M. malmoense,
M. marinum,
M. phlei,
M. scrofulaceum,
M. senegalense,
M. simiae,
M. smegmatis,
M. szulgai,
M. thermoresistibile,
M. triviale,
M. vaccae,
and M. xenopi.
Between 0.1 and 1 ng of
DNA was used as a template for each PCR assay,
to mimic the concentration
expected to occur in sputum
samples.
Expectorated sputum samples were collected from patients in Rio de
Janeiro who had a previous diagnosis of pulmonary tuberculosis
and who
were smear positive for multidrug-resistant
M. tuberculosis after 3 months of treatment. Drug resistance was confirmed by
conventional mycobacterial culture and susceptibility testing
(
6). The sputum samples were lysed and detoxified with
N-acetylcysteine
and NaOH (
9). One-milliliter
samples from 11 different patients
were brought to New York and spun in
a Shelton Scientific VSB-14
microcentrifuge (Shelton, Conn.) at 13,000 rpm for 5 min, and
each pellet was resuspended in 100 µl of 1 M NaOH
in 2% Triton
X-100. The samples were then boiled for 8 min and
neutralized
by the addition of 10 µl of 30% glacial acetic acid and
190 µl
of Tris-HCl (pH 8.0). DNA was extracted from each sample using
a Geneclean II kit (BIO 101, Carlsbad, Calif.), following the
manufacturer's instructions, and was then eluted into 20 µl of
H
2O. Five microliters of eluant was used as a
template for each
PCR
assay.
Assay conditions.
PCRs were performed in 96-well microtiter
plates (Applied Biosystems). Each well contained a total of 50 µl of
1× PCR buffer (Applied Biosystems), 4 mM MgCl2,
250 µM dATP, 250 µM dCTP, 250 µM dGTP, 250 µM dTTP, 2.5 U of
AmpliTaq Gold DNA polymerase (Applied Biosystems), a 500 nM
concentration of each primer, a 200 nM concentration of each
molecular beacon, and 5 µl of template DNA. The wells were
hermetically sealed prior to amplification to prevent
cross-contamination of untested samples. Amplification was performed in
an Applied Biosystems 7700 Prism spectrofluorometric thermal cycler.
The reaction mixtures were incubated for 10 min at 95°C to activate the DNA polymerase, followed by 40 to 50 cycles of 95°C denaturation for 30 s, 58°C annealing for 60 s, and 72°C extension for
30 s.
Fluorescence was measured in every well during each annealing step
throughout the course of each reaction. The spectral data
were
automatically analyzed by the computer program controlling
the
spectrofluorometric thermal cycler to determine the fluorescence
intensity contributed by each of the differently colored molecular
beacons. The background fluorescence of each probe (calculated
from the
readings that were taken between the 8th and the 15th
thermal cycles)
was then subtracted. These fluorescence intensities
were then
normalized to correct for intrinsic well-to-well variation
by
multiplying all of the fluorescence readings in each well by
a factor
that adjusted the maximum fluorescence intensity in each
well to the
same arbitrary value. The threshold cycle was automatically
determined
by the computer program controlling the spectrofluorometric
thermal
cycler to be the number of thermal cycles required before
the intensity
of the fluorescence signal exceeded six times the
standard deviation of
the
background.
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RESULTS |
Design of the assay.
Molecular beacons
(27) are oligonucleotides that contain a probe sequence
embedded within "arm" sequences that are unrelated to the probe.
The arms are chosen to be complementary to each other, so that under
assay conditions they hybridize to form a stem-and-loop secondary
structure in which the probe sequence is located in the loop. A
fluorophore is covalently linked to the end of one arm, and a
nonfluorescent quencher is covalently linked to the end of the other
arm. The fluorophore and the quencher are so close together in the stem
helix that fluorescence is suppressed (28). However, when
the probe binds to its complementary target, it forms a probe-target
helix that is longer and stronger than the stem helix. Since
double-stranded DNA is relatively rigid, it is topologically impossible
for the probe-target helix and the stem helix to coexist. Consequently,
the molecular beacon undergoes a conformational reorganization that
causes the arms to unwind. This separates the fluorophore from the
quencher, and the probe fluoresces brightly.
In order to detect any deletion, insertion, or nucleotide substitution
that may occur in the
rpoB core region, it was necessary
to
design the molecular beacons so that they each could hybridize
only to
the wild-type sequence. This was accomplished by choosing
short probe
sequences that form probe-target helices that are
just strong enough to
overcome the strength of the stem helix.
The presence of a mutation in
the target creates a mismatched
base pair that weakens the probe-target
helix, favoring the retention
of the stem helix. Because molecular
beacons can exist in two
different stable states (probe-target hybrid
or stem-and-loop
structure), they are "finicky" and form a
probe-target hybrid
only if the target is perfectly complementary to
the probe (
12,
28). Furthermore, it does not matter where
the mismatch occurs
within the probe-target hybrid. The melting
temperature of a mismatched
hybrid is significantly lower than the
melting temperature of
a perfectly complementary hybrid, irrespective
of the identity
or location of the mismatch (
1). Thus, the
presence of a mutation
anywhere within the
rpoB core region
will prevent the binding
of one of the molecular beacons, and the
characteristic fluorescence
of that molecular beacon will not
occur.
Five different molecular beacons were synthesized, each perfectly
complementary to a different segment of the wild-type
rpoB core. Together, the five molecular beacons covered the entire
core
sequence. In order to carry out the assay in a single tube,
each of the
molecular beacons was labeled with a differently colored
fluorophore.
Because five different colors needed to be detected
in the same assay,
it was highly desirable that each type of fluorophore
emit a signal of
comparable strength. However, we used a spectrofluorometric
thermal
cycler to carry out the assays that employs a blue argon-ion
laser to
stimulate fluorescence. Although energy from blue light
is well
absorbed by fluorophores that emit green or yellow fluorescence,
this
energy is not efficiently absorbed by fluorophores that emit
orange or
red fluorescence, resulting in relatively weak signals.
We therefore
used two conventional molecular beacons to generate
a green signal and
a yellow signal, and we used three "wavelength-shifting"
molecular
beacons to generate differently colored signals in the
orange-red
portion of the spectrum (
29). Figure
1 compares wavelength-shifting
and
conventional molecular beacons. In conventional molecular
beacons, a
single fluorophore carries out the dual functions of
absorbing energy
from the stimulating light and then emitting
that energy as fluorescent
light of a longer wavelength. In wavelength-shifting
molecular beacons,
two fluorophores are present on one arm of
the probe. A "harvester"
fluorophore efficiently absorbs energy
from the blue laser light, and
that energy is then rapidly transferred
to an "emitter" fluorophore
(by fluorescence resonance energy
transfer), which then fluoresces
brightly at its own characteristic
(orange or red) wavelength. Figure
2 shows the two complementary
strands of
the
rpoB core region and identifies the target sequence
for
each of the five molecular beacons.
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FIG. 1.
Comparison of conventional molecular beacons (top
diagrams) to wavelength-shifting molecular beacons (lower diagrams).
The fluorescence of both conventional and wavelength-shifting molecular
beacons is well quenched when the probes are free in solution (left
diagrams), yet both types of probes undergo a conformational
reorganization and fluoresce brightly when they bind to their target
(right diagrams). When a conventional molecular beacon is bound to a
target, its fluorophore absorbs energy from the stimulating light,
stores the energy for a few nanoseconds, and then emits that energy as
bright fluorescent light of a longer wavelength. However, if the
particular fluorophore that is chosen for a conventional molecular
beacon does not efficiently absorb energy from the stimulating light,
then its fluorescence signal will be weak (for example, when blue laser
light is used to stimulate the fluorescence of a red fluorophore). In
wavelength-shifting molecular beacons, however, the harvester
fluorophore is chosen because it efficiently absorbs energy from the
stimulating light (for example, fluorescein efficiently absorbs energy
from blue light), and the emitter fluorophore (usually orange or red)
is chosen because it is able to efficiently accept energy from the
harvester fluorophore, store the energy for a few nanoseconds, and then
emit that energy as bright fluorescent light at its own characteristic
wavelength.
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FIG. 2.
Location of the target sequence for each of the five
molecular beacons on the complementary strands of the 81-bp M.
tuberculosis rpoB core region. Probe B (labeled with
tetrachlorofluorescein) and Probe E (labeled with fluorescein)
were conventional molecular beacons, while Probe A (labeled with Texas
red), Probe C (labeled with tetramethylrhodamine), and Probe D (labeled
with rhodamine) were wavelength-shifting molecular beacons.
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The assay was designed to work as follows: DNA from a clinical sample
is amplified in the presence of the five differently
colored molecular
beacons. If all five fluorescent colors occur,
then
rifampin-susceptible
M. tuberculosis is present in the
sample;
if one or more probes fail to fluoresce, then
rifampin-resistant
M. tuberculosis is present; and if no
fluorescence occurs, then
M. tuberculosis is not present. In
addition, the assay was designed
to be sufficiently sensitive to detect
even a single bacillus
in a sputum sample; fluorescence measurements
are taken throughout
the course of the amplification, in order to
precisely determine
the number of bacilli in the
sample.
Detection of rifampin resistance.
Genomic DNA from 148 previously well-characterized M. tuberculosis clinical
isolates was tested. Conventional culture-based analysis had identified
83 of these isolates as being rifampin susceptible and 65 as being
rifampin resistant. Fluorescence signals developed in all 148 assays.
All five colors appeared in each of the 83 assays carried out with DNA
from the rifampin-susceptible isolates. Of the 65 rifampin-resistant
isolates that were tested, one color failed to develop in 59 isolates,
two colors failed to develop in four isolates (indicating the presence
of two different mutations), and two isolates developed all five
colors, despite being rifampin resistant. Subsequent nucleotide
sequence analysis of these two isolates showed that there were no
mutations in their rpoB core region. Thus, they were among
the 5% of rifampin-resistant clones whose resistance is due to a
mutation outside of the core region. The development of a multicolored
signal confirms that rpoB amplicons were synthesized. No
fluorescence developed in control reactions that did not contain
template DNA. Figure 3 shows
representative results obtained from two rifampin-susceptible isolates
and four rifampin-resistant isolates (one of which possessed two
mutations).
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FIG. 3.
Detection of mutations that cause rifampin resistance in
M. tuberculosis by the amplification of the
rpoB core region in the presence of five differently
colored molecular beacons. All of the probes hybridized to the
amplicons and generated strong fluorescence signals when
rifampin-susceptible strains were tested. However, one or two molecular
beacons failed to provide a fluorescence signal when rifampin-resistant
strains were tested. In all, 148 different M.
tuberculosis clinical isolates were tested. The results
obtained from six of these isolates are shown.
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Species specificity.
To determine whether the assay is
specific for M. tuberculosis, we tested DNA that was
isolated from 26 different mycobacterial species. The results (Fig.
4) show that only M. tuberculosis and the closely related M. africanum (a
member of the M. tuberculosis group, all of which cause
tuberculosis) elicited a positive response from each of the differently
colored rpoB molecular beacons that were present (the
fluorescein-labeled rpoB molecular beacon was purposely
omitted). Significantly, none of the rpoB molecular beacons
gave a positive signal with the other 24 species. To confirm that the
absence of these fluorescence signals was due to species-specific differences in the rpoB sequence, rather than being due to
PCR failure, each reaction was designed to generate an additional signal that served as an amplification control. The reactions contained
a second set of primers specific for a conserved region of the 16S rRNA
gene of mycobacteria and a fluorescein-labeled molecular beacon to
detect the resulting amplicon. This molecular beacon gave a signal with
all 26 species tested. Taken together, the results imply that positive
signals will occur only when M. tuberculosis or other
members of the M. tuberculosis group are present.
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FIG. 4.
Determination of the specificity of the assay.
Twenty-six mycobacterial species were tested (representative results
from eight of the species are shown). Only M.
tuberculosis (strain H37Rv) and the closely
related M. africanum (which is a member of the M.
tuberculosis group) elicited fluorescence from the four
differently colored rpoB probes that were present (a
fifth rpoB probe, labeled with fluorescein, was
omitted). The reactions also contained primers for the amplification of
a region of the 16S rRNA gene that is conserved in all mycobacteria and
a fluorescein-labeled molecular beacon that was complementary to that
region. The presence of fluorescence from the fluorescein-labeled probe
(plotted in green) served as a control signal that confirmed that the
absence of the other four colors was due to significant differences
between the rpoB sequence of M.
tuberculosis and the rpoB sequence in each of
the other species and not due to PCR failure.
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Sensitivity of the assay.
To determine the ability of the
assay to provide quantitative results, we prepared samples containing
10-fold serial dilutions of M. tuberculosis genomic DNA. The
most concentrated sample contained an amount of DNA equivalent to
2,000,000 bacilli, and the least concentrated sample contained an
amount of DNA equivalent to two bacilli. The lower the initial DNA
concentration, the greater the number of thermal cycles required to
generate a detectable fluorescence signal (the threshold cycle). The
results demonstrate (Fig. 5) that the
logarithm of the number of target DNA molecules initially present is
inversely proportional to the threshold cycle (8). The
assay is thus quantitative over an extremely wide range of target
concentrations, and it is sufficiently sensitive to detect the DNA from
two bacilli.
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FIG. 5.
Determination of the sensitivity of the assay. Eight PCR
assays were initiated with different quantities of DNA obtained from
the M. tuberculosis laboratory strain,
H37Rv. The amount of DNA added as a template to each of
the eight reactions was calculated to be equivalent to the amount of
genomic DNA contained in 0, 2, 20, 200, 2,000, 20,000, 200,000, and
2,000,000 bacilli, respectively. Primers were present to amplify the
rpoB core region, and probe E was used to detect the
amplicons in real time. The results (shown in the left panel)
demonstrate that the number of amplification cycles required to
generate a detectable fluorescence signal decreases as the number of
target molecules initially present in a reaction increases. A control
reaction, which did not contain any template DNA, did not give a
signal. The results (shown in the right panel) demonstrate that: (i)
the threshold cycle is inversely proportional to the logarithm of the
number of target molecules initially present, (ii) quantitative results
can be obtained over a wide range of target concentrations, and (iii)
the assay is sufficiently sensitive to detect as few as two bacilli.
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When preparing samples by serial dilution, it is difficult to obtain a
sample containing exactly two genomic DNA equivalents.
A test reaction
can, by chance, contain a greater or smaller number
of DNA molecules.
We therefore prepared 10 different samples designed
to contain two
genomic DNA equivalents and found that 8 of the
10 samples gave
positive signals. In addition, we prepared 10
different samples
designed to contain two-tenths of a genomic
DNA equivalent and found
that 1 of the 10 samples gave a positive
signal. These results imply
that the assay can detect a single
target molecule if it is present in
the
sample.
Determination of rifampin susceptibility in sputum samples.
DNA was extracted from 11 smear-positive sputum samples obtained from
patients infected with rifampin-resistant M. tuberculosis and was used as a template in the assay. A control sample containing DNA extracted from a rifampin-susceptible M. tuberculosis
laboratory strain was evaluated in parallel. All five differently
colored molecular beacons gave a fluorescence signal with the
rifampin-susceptible control, while one of the five differently colored
molecular beacons failed to give a signal with each of the 11 rifampin-resistant samples (Fig. 6).
Subsequent nucleotide sequence analysis of the rpoB core
region of the amplicons generated from each sputum sample showed that a
mutation was present within the target sequence of whichever molecular
beacon failed to produce a fluorescence signal.
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FIG. 6.
Determination of the rifampin susceptibility of
M. tuberculosis found in sputum samples. Eleven PCR
assays were carried out with DNA extracted from sputum obtained from
smear-positive patients infected with rifampin-resistant M.
tuberculosis. A control reaction contained DNA from the
rifampin-susceptible M. tuberculosis laboratory strain,
H37Rv. Although all five differently colored molecular
beacons gave a fluorescence signal with the rifampin-susceptible
control, one of the five fluorescent colors failed to develop in each
of the 11 rifampin-resistant samples.
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DISCUSSION |
The results demonstrate that rifampin-resistant M. tuberculosis can be detected in DNA isolated from sputum samples
in a single-tube assay that takes less than 3 h to perform. The
assay is extremely specific and extraordinarily sensitive. Moreover,
the assay is simple to perform and readily automatable for
high-throughput screening. The results that are obtained from the assay
indicate whether a patient is infected with M. tuberculosis,
what concentration of bacilli is present in the sample, and whether the
bacilli are rifampin resistant. Because all multidrug-resistant
M. tuberculosis strains are rifampin resistant
(23), the results of the assay enable an immediate
decision to be made as to whether to prescribe a more rigorous course
of antibiotic treatment. Furthermore, the assay provides quantitative
results, which could eliminate the need to perform sputum microscopy
for routine tuberculosis screening.
We utilized wavelength-shifting molecular beacons to provide strong
fluorescence signals in the orange and red portion of the visible
spectrum. This was necessitated by our use of the Applied Biosystems
7700 spectrofluorometric thermal cycler, which employs a blue argon-ion
laser to stimulate fluorescence. However, a number of other instruments
are capable of carrying out multiplex real-time gene amplification
assays utilizing multicolored light sources for stimulating
fluorescence (for example, the Bio-Rad iCycler iQ, the Cepheid Smart
Cycler, and the Stratagene Mx4000). All of the probes used in the assay
can be conventional molecular beacons if one of these instruments is
used. Although the spectrofluorometric instruments used to carry out
these assays are relatively expensive, they are becoming common in
clinical diagnostic laboratories as a result of an increasing awareness
of the broad applicability of closed-tube gene amplification techniques
(20, 25, 30).
It should also be possible to replace the multitemperature PCR that is
used in the assay with an isothermal gene amplification protocol. For
example, molecular beacons provide strong signals with amplicons
produced during nucleic acid sequence-based amplification (10), rolling-circle amplification (11, 32),
and strand-displacement amplification (31).
Commercial versions of the assay should include a sixth uniquely
colored probe that provides a positive signal to confirm that gene
amplification is taking place. Ultimately, these assays will enable
more rapid diagnosis, earlier treatment, and prompt implementation of
infection control procedures to reduce the morbidity, mortality, and
spread of drug-resistant tuberculosis.
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ACKNOWLEDGMENTS |
We thank Richard Chaisson of Johns Hopkins University in
Baltimore, Md., and Fernanda Mello and Afranio Kritski of the
University of Rio de Janeiro in Brazil for providing infected sputum
samples, Amalio Telenti of the Centre Hospitalier Universitaire Vaudois in Switzerland and Michael Levi of Montefiore Medical Center for providing cultured M. tuberculosis isolates, and Pablo
Bifani and Barry Kreiswirth of the Public Health Research Institute for providing purified DNA from other mycobacterial species. We also thank
Cindy Fung for her expert technical assistance.
This work was supported by National Institutes of Health grants
AI-07501, AI-43268, AI-46669, and HL-43521.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics, Public Health Research Institute, 455 First Ave., New York, NY 10016. Phone: (212) 578-0870. Fax: (212) 576-8471. E-mail:
kramer{at}phri.nyu.edu.
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REFERENCES |
| 1.
|
Bonnet, G.,
S. Tyagi,
A. Libchaber, and F. R. Kramer.
1999.
Thermodynamic basis of the enhanced specificity of structured DNA probes.
Proc. Natl. Acad. Sci. USA
96:6171-6176[Abstract/Free Full Text].
|
| 2.
|
De Beenhouwer, H.,
Z. Lhiang,
G. Jannes,
W. Mijs,
L. Machtelinckx,
R. Rossau,
H. Traore, and F. Portaels.
1995.
Rapid detection of rifampin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay.
Tuber. Lung Dis.
76:425-430[CrossRef][Medline].
|
| 3.
|
Fischl, M. A.,
R. B. Uttamchandani,
G. L. Daikos,
R. B. Poblete,
J. N. Moreno,
R. R. Reyes,
A. M. Boota,
L. M. Thompson,
T. J. Cleary, and S. Lai.
1992.
An outbreak of tuberculosis caused by multiple-drug-resistant tubercle bacilli among patients with HIV infection.
Ann. Intern. Med.
117:177-183.
|
| 4.
|
Goble, M.,
M. D. Iseman,
L. A. Madsen,
D. Waite,
L. Ackerson, and C. R. Horsburgh, Jr.
1993.
Treatment of 171 patients with pulmonary tuberculosis resistant to isoniazid and rifampin.
N. Engl. J. Med.
328:527-532[Abstract/Free Full Text].
|
| 5.
|
Harris, G.,
A. Rayner,
J. Blair, and B. Watt.
2000.
Comparison of three isolation systems for the culture of mycobacteria from respiratory and non-respiratory samples.
J. Clin. Pathol.
53:615-618[Abstract/Free Full Text].
|
| 6.
|
Heifets, L. B.
1986.
Rapid automated methods (BACTEC system) in clinical mycobacteriology.
Semin. Respir. Infect.
1:242-249[Medline].
|
| 7.
|
Heifets, L. B., and G. A. Cangelosi.
1999.
Drug susceptibility testing of Mycobacterium tuberculosis: a neglected problem at the turn of the century.
Int. J. Tuberc. Lung Dis.
3:564-581[Medline].
|
| 8.
|
Higuchi, R.,
C. Fockler,
G. Dillinger, and R. Watson.
1993.
Kinetic PCR analysis: real-time monitoring of DNA amplification reactions.
Bio/Technology
11:1026-1030[CrossRef][Medline].
|
| 9.
|
Kent, P. R., and G. P. Kubica.
1985.
Public health mycobacteriology: a guide for the level III laboratory. U.S.
Department of Health and Human Services, Washington, D.C.
|
| 10.
|
Leone, G.,
H. van Schijndel,
B. van Gemen,
F. R. Kramer, and C. D. Schoen.
1998.
Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA.
Nucleic Acids Res.
26:2150-2155[Abstract/Free Full Text].
|
| 11.
|
Lizardi, P. M.,
X. Huang,
Z. Zhu,
P. Bray-Ward,
D. C. Thomas, and D. C. Ward.
1998.
Mutation detection and single-molecule counting using isothermal rolling-circle amplification.
Nat. Genet.
19:225-232[CrossRef][Medline].
|
| 12.
|
Marras, S. A. E.,
F. R. Kramer, and S. Tyagi.
1999.
Multiplex detection of single-nucleotide variations using molecular beacons.
Genet. Anal.
14:151-156[Medline].
|
| 13.
|
Mitchison, D. A., and A. J. Nunn.
1986.
Influence of initial drug resistance on the response to short-course chemotherapy of pulmonary tuberculosis.
Am. Rev. Respir. Dis.
133:423-430[Medline].
|
| 14.
|
Musser, J. M.
1995.
Antimicrobial agent resistance in mycobacteria: molecular genetic insights.
Clin. Microbiol. Rev.
8:496-514[Abstract].
|
| 15.
|
Pablos-Mendez, A.,
T. R. Sterling, and T. R. Frieden.
1996.
The relationship between delayed or incomplete treatment and all-cause mortality in patients with tuberculosis.
JAMA
276:1223-1228[Abstract/Free Full Text].
|
| 16.
|
Pablos-Mendez, A.,
M. C. Raviglione,
A. Laszlo,
N. Binkin,
H. L. Rieder,
F. Bustreo,
D. L. Cohn,
C. S. Lambregts-van Weezenbeek,
S. J. Kim,
P. Chaulet, and P. Nunn.
1998.
Global surveillance for antituberculosis-drug resistance, 1994-1997.
N. Engl. J. Med.
338:1641-1649[Abstract/Free Full Text].
|
| 17.
|
Palacios, J. J.,
J. Ferro,
N. Ruiz Palma,
J. M. García,
H. Villar,
J. Rodríguez,
M. D. Macías, and P. Prendes.
1999.
Fully automated liquid culture system compared with Löwenstein-Jensen solid medium for rapid recovery of mycobacteria from clinical samples.
Eur. J. Clin. Microbiol. Infect. Dis.
18:265-273[CrossRef][Medline].
|
| 18.
|
Piatek, A. S.,
S. Tyagi,
A. C. Pol,
A. Telenti,
L. P. Miller,
F. R. Kramer, and D. Alland.
1998.
Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis.
Nat. Biotechnol.
16:359-363[CrossRef][Medline].
|
| 19.
|
Piatek, A. S.,
A. Telenti,
M. R. Murray,
H. El-Hajj,
W. R. Jacobs, Jr.,
F. R. Kramer, and D. Alland.
2000.
Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing.
Antimicrob. Agents Chemother.
44:103-110[Abstract/Free Full Text].
|
| 20.
|
Pierce, K. E.,
J. E. Rice,
J. A. Sanchez,
C. Brenner, and L. J. Wangh.
2000.
Real-time PCR using molecular beacons for accurate detection of the Y chromosome in single human blastomeres.
Mol. Hum. Reprod.
6:1155-1164[Abstract/Free Full Text].
|
| 21.
|
Riska, P. F.,
W. R. Jacobs, Jr., and D. Alland.
2000.
Molecular determinates of drug resistance in tuberculosis.
Int. J. Tuberc. Lung Dis.
4:S4-S10[Medline].
|
| 22.
|
Sreevatsan, S.,
X. Pan,
K. E. Stockbauer,
N. D. Connell,
B. N. Kreiswirth,
T. S. Whittam, and J. M. Musser.
1997.
Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination.
Proc. Natl. Acad. Sci. USA
94:9869-9874[Abstract/Free Full Text].
|
| 23.
|
Telenti, A.,
P. Imboden,
F. Marchesi,
D. Lowrie,
S. Cole,
M. J. Colston,
L. Matter,
K. Schopfer, and T. Bodmer.
1993.
Detection of rifampin-resistance mutations in Mycobacterium tuberculosis.
Lancet
341:647-650[CrossRef][Medline].
|
| 24.
|
Telenti, A.,
N. Honore,
C. Bernasconi,
J. March,
A. Ortega,
B. Heym,
H. E. Takiff, and S. T. Cole.
1997.
Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at a reference laboratory level.
J. Clin. Microbiol.
35:719-723[Abstract].
|
| 25.
|
Torres, M. J.,
A. Criado,
J. C. Palomares, and J. Aznar.
2000.
Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis.
J. Clin. Microbiol.
38:3194-3199[Abstract/Free Full Text].
|
| 26.
|
Turett, G. S.,
E. E. Telzac,
L. V. Torian,
S. Blum,
D. Alland,
I. Weisfuse, and B. A. Fazal.
1995.
Improved outcomes for patients with multidrug-resistant tuberculosis.
Clin. Infect. Dis.
21:1238-1244[Medline].
|
| 27.
|
Tyagi, S., and F. R. Kramer.
1996.
Molecular beacons: probes that fluoresce upon hybridization.
Nat. Biotechnol.
14:303-308[CrossRef][Medline].
|
| 28.
|
Tyagi, S.,
D. P. Bratu, and F. R. Kramer.
1998.
Multicolor molecular beacons for allele discrimination.
Nat. Biotechnol.
16:49-53[CrossRef][Medline].
|
| 29.
|
Tyagi, S.,
S. A. E. Marras, and F. R. Kramer.
2000.
Wavelength-shifting molecular beacons.
Nat. Biotechnol.
18:1191-1196[CrossRef][Medline].
|
| 30.
|
Vet, J. A. M.,
A. R. Majithia,
S. A. E. Marras,
S. Tyagi,
S. Dube,
B. Poiesz, and F. R. Kramer.
1999.
Multiplex detection of four pathogenic retroviruses using molecular beacons.
Proc. Natl. Acad. Sci. USA
96:6394-6399[Abstract/Free Full Text].
|
| 31.
|
Walker, G. T.,
M. S. Fraiser,
J. L. Schram,
M. C. Little,
J. G. Nadeau, and D. P. Malinowski.
1992.
Strand displacement amplification an isothermal, in vitro DNA amplification technique.
Nucleic Acids Res.
20:1691-1696[Abstract/Free Full Text].
|
| 32.
|
Zhang, D. Y.,
M. Brandwein,
T. C. Hsuih, and H. Li.
1998.
Amplification of target-specific, ligation-dependent circular probe.
Gene
211:277-285[CrossRef][Medline].
|
Journal of Clinical Microbiology, November 2001, p. 4131-4137, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4131-4137.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
