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Journal of Clinical Microbiology, September 2000, p. 3194-3199, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Use of Real-Time PCR and Fluorimetry for Rapid
Detection of Rifampin and Isoniazid Resistance-Associated Mutations
in Mycobacterium tuberculosis
Maria J.
Torres,*
Antonio
Criado,
Jose C.
Palomares, and
Javier
Aznar
Unidad de Microbiología Molecular,
Departamento de Microbiología, Universidad de Sevilla, 41080 Seville, Spain
 |
ABSTRACT |
Very fast amplification of DNA in small volumes can be continuously
monitored with a rapid cycler that incorporates fluorimetric detection.
Primers were designed to amplify a 157-bp fragment of the
rpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium
tuberculosis. Most mutations associated with resistance to
rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these
codons. Two pairs of hybridization probes were synthesized; one in each
pair was 3' labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5' labeled with
LightCycler-Red 640. Each pair of probes recognized adjacent sequences
in the amplicon. After DNA amplification was finished by using a
LightCycler, the temperature at which the Red 640 probe melted from the
product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH,
RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to
both antimicrobial agents) were amplified, and the presence of
mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the
LightCycler method correctly genotyped all the strains without the need
of any post-PCR sample manipulation. Overall, this pilot study
demonstrated that real-time PCR coupled to fluorescence detection is
the fastest available method for the detection of RMP and INH
resistance-associated mutations in M. tuberculosis clinical isolates.
| |
INTRODUCTION |
The emergence of drug-resistant
strains of Mycobacterium tuberculosis is an increasing
problem for populations and tuberculosis control programs in developed
and developing countries alike. Today, rifampin (RMP) and isoniazid
(INH) are important components of effective multidrug therapy and
prophylaxis for M. tuberculosis infections. However,
widespread use of these agents and failure of patients to complete
prescribed treatment have led to the emergence of RMP- and
INH-resistant strains (5, 7). RMP resistance is well
characterized, and 95% of all M. tuberculosis RMP-resistant clinical isolates harbor specific mutations within an 81-bp region of
the rpoB gene, which encodes the 
subunit of the RNA
polymerase (8, 18). In contrast to RMP, genotypic testing
for INH resistance is much more complex. Several studies have reported
alterations in at least four genes, katG, inhA,
ahpC, and kasA (1, 10, 12, 21, 24).
Although the importance of katG mutations in INH resistance
has been clearly demonstrated, the involvement of the other genes is
arguable (6, 10, 11, 15). The highest proportion of
mutations is in the katG gene, but only some mutations in
this gene have been associated with high-level resistance (e.g., deletions or S315T), whereas others do not appear to confer any resistance to INH (e.g., R463L).
The need to minimize the transmission of drug-resistant strains
requires rapid identification procedures. Due to their high sensitivity
and specificity, DNA assays have the potential to provide rapid
detection of resistance in mycobacterial isolates. Because these
procedures do not rely on in vitro growth, the time between diagnosis
and the onset of effective therapy is shortened.
In this report we present a single-tube method for detecting mutations
associated with resistance to RMP and INH. It combines both rapid-cycle
PCR and real-time monitoring of the processing and generation of
mutation-specific, fluorescent-probe melting profiles on the
LightCycler (Roche Biochemicals). Two fluorescently labeled
hybridization probes recognizing adjacent sequences in the amplicon
were present in the reaction mixture. The shorter detection probe
(sensor [5'-LightCycler-Red 640 labeled]) covers the predicted site
of mutation, while the longer probe (anchor [3' fluorescein labeled])
produces the fluorescent signal. After annealing, the fluorophores are
in close proximity and there can be a fluorescence resonance energy
transfer between them, providing real-time monitoring of the
amplification process. When PCR was completed, fluorescence was
monitored as the temperature was increased through the melting
temperature (Tm) of the probe-product duplexes, and a characteristic melting profile for each genotype was obtained. We
used two different pairs of probes
one designed to detect the two most
frequent mutations, Ser531Leu (a change in codon 531 from coding serine
to leucine [TCG
TTG]) and His526Asp (a change in codon 526 from
coding histidine to aspartic acid [CACGAC]) in the rpoB
gene related to RMP resistance, and the other designed to detect the
most frequent mutation (in codon 315) related to INH resistance in the
katG gene. With this method one strain could be genotyped
within 30 min without any post-PCR sample manipulation.
| |
MATERIALS AND METHODS |
Strains and resistance testing.
Twenty susceptible and 36 resistant clinical isolates of M. tuberculosis (16 resistant
to RMP, 16 to INH, and 4 to both), as determined by the radiometric
dilution method (23), from 56 different patients were
studied. By this method, drug resistance was defined as greater than
1% growth in the presence of 2 µg of streptomycin per ml, 0.2 µg
of INH per ml, 2 µg of RMP per ml, or 5 µg of ethambutol per ml.
Isolates that were found to be resistant by this method were retested
by the proportion method before being reported as resistant
(23).
Relatedness of all resistant strains was investigated by restriction
fragment length polymorphism of the IS6110 element
(16). All the strains had different IS6110
fingerprint patterns. M. tuberculosis H37Rv (susceptible),
M. tuberculosis ATCC 35838 (RMP resistant), and M. tuberculosis ATCC 35822 (INH resistant) were used as controls.
Extraction of mycobacterial DNA from the strains.
A rapid
DNA extraction procedure for direct testing of M. tuberculosis on Löwenstein-Jensen solid medium was performed
as follows. One 10-µl loop of the organisms was suspended in 100 µl
of sterile water, and subsequently 100 µl of a 10% suspension of
Chelex 100 was added (20). After thorough mixing, the
mixture was incubated at 45°C for 45 min, then the suspension was
boiled for 5 min, and the bacterial debris was removed by
centrifugation (12,000 × g for 5 min). The supernatant
was directly used for amplification.
Conventional PCR.
The DNA preparation was amplified with the
primers listed in Table 1. PCR primers
were designed to amplify the regions between nucleotides 2335 and 2492 of the rpoB gene and nucleotides 2759 and 2967 of the
katG gene (19). Conditions for cycling were 95°C for 45 s followed by 35 cycles of 95°C for 15 s,
55°C (annealing temperature for the rpoB gene) and 60°C
(annealing temperature for the katG gene) for 15 s, and
72°C for 15 s in a 9600 Perkin Elmer apparatus. PCR products
were purified by using the Sephaglas BandPrep kit (Amersham Pharmacia
Biotech).
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TABLE 1.
Oligonucleotidesprimersa and
probesused in the amplification and detection protocol by PCR of the
M. tuberculosis strains
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|
PCR-single-strand conformation polymorphism analysis
(3).
After amplification by the same PCR protocol as
above, 50 ng of the purified PCR product was mixed 1:1 with at least 2 µl of loading buffer (99% formamide-5 mM EDTA-0.5% bromophenol
blue), heated for 10 min at 95°C, cooled on ice, and loaded in a
precast, ready-to-use gel (Genegel Excel 12,5/24 kit; Amersham
Pharmacia Biotech). Electrophoresis was run on a GenePhor
electrophoresis unit at 600 V, 25 mA, 15 W, and 15°C for about 80 min
(until bromophenol blue reached the anode buffer strip). Gels were
stained in a Hoefer automated gel stainer with PlusOne DNA silver stain
(Amersham Pharmacia Biotech). This method was used as part of the
characterization of resistant strains.
DNA sequencing.
Direct sequencing of PCR products of all the
strains was performed with a LiQor automated sequencer and
corresponding kits from the same manufacturer (Roche Biochemicals).
LightCycler.
All DNA extracts were amplified with the
primers used in conventional PCR, with the addition of the fluorescein-
and Red 640-labeled probes to the amplification mix. The probes were
designed as shown in Fig. 1. For
rpoB the 5'-Red 640-labeled probe (rpo sensor) covered the region containing codons 526 and 531. To avoid dimer formation between primer TR8 and the rpo anchor probe, a
GT substitution at position 2407 (GenBank accession number L27989)
was introduced in the 3' terminus of the rpo anchor probe.
There was a four-base gap between the rpo sensor and the
3'-fluorescein-labeled probe (rpo anchor). The theoretical
Tms were 61 and 68.4°C for TR9 and TR8
primers, respectively, and 70.4 and 73.8°C for the rpo
sensor and rpo anchor probes, respectively. For the
katG gene, the 5'-Red 640-labeled probe (katG
sensor) covered the region containing codon 315. There was a two-base
gap between the katG sensor and the 3'-fluorescein-labeled
probe (katG anchor). The theoretical Tms were 64.3 and 62.8°C for TB86 and TB87
primers, respectively, and 69.4 and 75.3°C for katG sensor
and katG anchor probes, respectively (Table 1). Thus, the
Red 640-labeled probes should melt from the PCR product at a lower
temperature than the fluorescein-labeled probes so that the
fluorescence observed is a measure of the Red 640-labeled probes. All
primers and probes were designed and synthesized by TIB MOLBIOL (DNA
Synthesis Service; Roche Diagnostics, Berlin, Germany).
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FIG. 1.
Design of fluorescent probes for mutation detection in
the rpoB (A) and katG (B) genes in the
LightCycler. The probes are designed so that the Red 640-labeled probe
will detach at a lower temperature than the fluorescein-labeled probe.
Primers were those described by Telenti et al. (19).
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|
The components for PCR in a final volume of 20 µl included 2 µl of
a commercial ready-to-use reaction mix for PCR (LightCycler-DNA
master
hybridization probes; Roche Diagnostics) that contains
Taq
DNA polymerase, reaction buffer, deoxynucleoside triphosphate
mix, and
10 mM MgCl
2. We added MgCl
2 to a final
concentration
of 4 mM. The primers and probes were added to final
concentrations
of 0.5 and 0.2 µM, respectively. Finally, we used 2 µl of template
DNA. The 20-µl (final volume) reaction mix was
placed in glass
capillary cuvettes which were filled by pulse
centrifugation in
a microcentrifuge. Conditions for cycling were 95°C
for 45 s,
followed by 35 cycles of 95°C for 1 s, 55°C
(annealing temperature
for the
rpoB gene) and 60°C
(annealing temperature for the
katG gene) for 2 s, and
72°C for 6 s, with monitoring of fluorescence
during the
annealing phase; this, in turn, was followed by a melting
program of 50 to 85°C at 0.1°C/s with continuous monitoring of
the
fluorescence.
| |
RESULTS |
Rapid PCR and continuous monitoring of product.
The
accumulation of PCR product was monitored by measuring the level of
fluorescence. Figure 2 shows the progress
of PCR with Chelex 100-extracted DNA as the template. The PCR product
was observed to accumulate in an exponential manner, indicating an optimal PCR. It can be seen that the signals started to rise at different times, usually between cycle numbers 15 and 25, because we
did not estimate the DNA concentration and started with different amounts of DNA. This did not affect the interpretation of the Tm. We used the same primers for
"conventional" PCR and LightCycler, because our target was small
enough that this rapid cycling protocol worked well.
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FIG. 2.
Accumulation of Red 640 fluorescence during PCR using
the katG anchor and sensor probes with three different
samples: M. tuberculosis H37Rv strain (susceptible) and R27
and R30 M. tuberculosis strains (INH resistant). NC,
negative control. F2 is the channel used by the LightCycler to detect
the LightCycler-Red 640 light emission.
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Tms of the probes.
The temperatures at
which the probes melted from PCR products during the melting program
were calculated using the LightCycler software. A summary of the
Tms for all of the probes (RMP and INH) together
with the changes in Tm for the products derived from resistant and susceptible M. tuberculosis strains is
shown in Table 2.
We were able to detect the two most frequent mutations responsible for
RMP resistance, using the RMP probe (
rpo sensor) (Fig.
3). The
Tm for the
susceptible strain used as a control (
M. tuberculosis H37Rv)
was 64.3°C, while the change from wild type to mutant at
codon 531 (TCG
TTG) in 18 strains resulted in a >2°C increase
in the
probe's
Tm. In the two strains with mutations
at codon
526 (CAC
GAC), we observed a >6°C drop in the probe's
Tm.
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FIG. 3.
Derivative melting curves (dF/dT) of Red 640-labeled
rpo sensor probe with three different samples. This sensor
probe was designed to have the sequence CTG at codon 531 and the
wild-type sequence (CAC) at codon 526. Strains: H3TRv, M. tuberculosis H37Rv strain (susceptible) with a double mismatch at
codon 531 (TCG) resulting in a Tm of 64.4°C;
R4, M. tuberculosis R4 strain (RMP resistant) with a
531-codon point mutation (TCG to TTG), which has a higher
Tm of 66.9°C showing a single mismatch; R15,
M. tuberculosis R15 strain (RMP resistant) with a 526-codon
point mutation (CAC to GAC), which has the lower
Tm due to the three mismatches, one at codon 526 and two at codon 531. Each trace shows the change in fluorescence ratio
with time with respect to temperature, thus allowing calculation of the
temperature at which the probe detaches from the PCR product. F2 is the
channel used by the LightCycler to detect the LightCycler-Red 640 light
emission; F1 is the channel measuring the background.
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Figure
4 shows the results with the INH
probe (
katG sensor). The
Tm for the
susceptible strain (
M. tuberculosis H37Rv) was
72.8°C. The
change from wild type (AGC) to ACC at codon 315 in
18 strains resulted
in a >3°C drop of
Tm of the
katG
sensor probe.
A drop of almost 5°C in the
Tm
was found in two strains harboring
a different mutation at codon 315 (AGC
AAC).
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FIG. 4.
Derivative melting curves of Red 640-labeled
katG sensor probe with three different samples: M. tuberculosis H37Rv strain (susceptible) with a perfect match to
the hybridization probe and a Tm of 72.8°C;
M. tuberculosis strain R30 (INH resistant) with the mutation
AGC ACC at codon 315, and a Tm of 68.7°C;
and M. tuberculosis strain R27 with the mutation AGC AAC
at codon 315 and a Tm of 67.9°C. Each trace
shows the change in fluorescence ratio over time with respect to
temperature, thus allowing calculation of the temperature at which the
probe detaches from the PCR product. F2 is the channel used by the
LightCycler to detect LightCycler-Red 640 light emission; F1 is the
channel measuring the background.
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All the susceptible
M. tuberculosis strains, determined by
using either the
rpo sensor or the
katG sensor
probe, showed a
Tm that corresponded to the
susceptible strain used as control
(
M. tuberculosis H37Rv).
In addition, the sequence inferred from
the LightCycler data was always
in accordance with the nucleotide
sequencing and the single-strand
conformational polymorphism
pattern.
| |
DISCUSSION |
Due to the slow growth of the M. tuberculosis bacillus,
unacceptable delays in the diagnosis of drug-resistant tuberculosis can
occur when conventional culture-based susceptibility tests are used.
Understanding the genetic events that lead to drug resistance in
clinical M. tuberculosis isolates is important for
elucidation of the action of antimicrobial agents, for design of novel
antibiotics that are active against drug-resistant strains, and for
development of genetic assays to be adapted to the routine clinical
mycobacteriology laboratory. Rapid approaches that use genetic analysis
for detection of mutations associated with drug resistance have been
proposed (4, 9, 13, 15, 19). However, these methods have
restricted applicability in clinical laboratories and are mainly
limited to the detection of RMP resistance.
The method described here provides a rapid, accurate, and inexpensive
way to detect resistance in M. tuberculosis clinical isolates. Amplification, hybridization, and analysis are all performed simultaneously in sealed capillary tubes. The entire assay, including analysis, can be completed in 30 min. In all cases, the result obtained
with the LightCycler corresponded to nucleotide sequence data;
therefore, unlike many other nucleic amplification tests (14), the LightCycler amplification assay was 100%
specific. This was likely due to the closed-well protocol, which
eliminates amplicon carryover. Theoretically, it is possible that
natural variability in the sequences where the sensor probes bind could lead to a change in the Tm of the probe that is
not associated with resistance mutation. However, previous studies
(17) have suggested that the DNA sequences of M. tuberculosis are extraordinarily well conserved and that mutations
in the M. tuberculosis genome are almost always associated
with drug resistance. This fact makes this method even more useful for
resistance detection in M. tuberculosis.
With the RMP probe used in this study, we were able to detect mutations
at codon 531 and codon 526. We previously reported (6) that
these were the two most frequent residue changes associated with RMP
resistance in our study area, and this finding was similar to previous
studies (8, 18). Based on our mutation data from strains
isolated in the last 6 years (6; unpublished data), if we used an additional probe covering codons 513 and 518, the sensitivity for the detection of RMP resistance would be greater than
95%.
In contrast to RMP resistance testing, genotypic testing for resistance
to INH presents many difficulties. Our understanding of the genetic
basis of INH resistance is far from complete. Changes in the
katG gene, the inhA gene, and the ahpC
gene in INH-resistant mycobacterial isolates have been described. The
importance of katG mutations in INH resistance is well
established, although the extent to which such mutations account for
the spectrum of resistance is still debated. Missense mutations at
codon 315 of the katG gene have been reported in almost
two-thirds of the INH-resistant isolates from different geographic
areas including ours (6, 13, 19). The high percentage of
mutations at position 315 and the occurrence of different nonsynonymous
substitutions demonstrate the importance of this codon for the
development of INH resistance among M. tuberculosis strains.
Mutations at other positions seem to be rare events. In addition, many
of these do not alter enzyme activity, and usually it is suggested that
they are involved in the resistance mechanism simply because no other
katG gene mutations were observed. For all these reasons, we
decided to assess the utility of this new method by using a probe to
screen for the presence of mutation in a selected region of the
katG gene that includes position 315. The specificity of the
test was 100%, and the Tm of the probe allowed
the two mutations studied to be distinguished.
We are working on the development of a probe to screen for mutations in
the regulatory region of the inhA gene that cause overexpression of this gene. However, in our sample population, we
found this alteration (nucleotide substitution at position 209 [CT]) in only 10% of the strains, and there is still some controversy about the implication of this gene in INH resistance (11).
The presence of mutations in either of these two genes (i.e.,
katG or inhA) would account for INH resistance,
but, according to previous studies on different populations
(15), between 20 and 60% of resistant strains have no
detectable mutations in those genes. Therefore, more research is needed
to elucidate the remaining cases of resistant strains that cannot be
attributed to the above-mentioned mutations in katG and
inhA genes. Furthermore, in both RMP and INH resistance, the
relative frequency of particular (i.e., rare) mutations may reflect
local strain populations and/or epidemic strains. Thus, determination
of the frequency of mutations underlying drug resistance in isolates
from different geographic areas has to be the basis for developing
rapid and specific techniques for the detection of drug-resistant
strains by molecular genetic techniques.
This new method presents several advantages. First, rapid amplification
and analysis allow the test to be completed within 30 min. Because the
DNA extraction procedure requires 1 h, the whole detection can be
completed in less than 2 h. Second, the LightCycler optical device
is capable of measuring fluorescence in two separate channels
simultaneously (Red 640 and Red 705 Fluorophore), and it is possible to
use Sybr Green I as a generic donor of fluorescence resonance energy
transfer (2, 22) (instead of a specific fluorescein probe),
thus allowing analysis of different mutations within a single test
tube. Third, the assay is run in closed glass capillaries.
Postamplification analysis can be performed without opening the
capillaries, minimizing the risk of carryover contamination. Fourth,
the LightCycler could be used to determine the genetic make-up of some
bacterial populations composed of subpopulations with different
genotypes. Fifth, the method can be adapted to genotyping resistance
information emerging from the identification of additional targets,
particularly for INH resistance.
Finally, we emphasize that this is the first time that real-time PCR
has been employed to study resistance in M. tuberculosis clinical isolates. Reliable results can be obtained faster than with
any other current molecular methods and could be directly applied to
clinical samples.
| |
ACKNOWLEDGMENT |
This study was financially supported by grant 99/0269 from the
Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo, Madrid, Spain.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Microbiología, Facultad de Medicina, Apdo 914, 41080 Seville,
Spain. Phone: 34-54552862. Fax: 34-54377413. E-mail:
folia{at}cica.es.
| |
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Journal of Clinical Microbiology, September 2000, p. 3194-3199, Vol. 38, No. 9
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
