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Journal of Clinical Microbiology, October 2000, p. 3656-3662, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Genotypic Determination of Mycobacterium tuberculosis
Antibiotic Resistance Using a Novel Mutation Detection Method, the
Branch Migration Inhibition M. tuberculosis Antibiotic
Resistance Test
Y. P.
Liu,1,*
M. A.
Behr,2
P. M.
Small,2 and
N.
Kurn1
Advanced Diagnostics Division, Dade Behring
Inc., San Jose,1 and Department of
Medicine, Stanford University Medical Center, Palo
Alto,2 California
Received 19 April 2000/Returned for modification 1 June
2000/Accepted 31 July 2000
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ABSTRACT |
A novel method for the detection of any alteration within a defined
sequence has recently been demonstrated (A. Lishanski, N. Kurn, and
E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin.
Chem. 46:9, 2000). Essential to this method are the generation of
partial duplexes that are capable of forming four-stranded structures
and the ability to detect inhibition of branch migration in these
structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol.
230:413-424, 1993). Inhibition of branch migration indicates the
presence of sequence alteration. This mutation detection method, termed
branch migration inhibition (BMI), is suitable for the detection of
drug resistance in M. tuberculosis, which is frequently
associated with multiple mutations within known genes. We describe the
genotypic determination of the rifampin (RMP) and pyrazinamide (PZA)
susceptibilities of M. tuberculosis isolates, using BMI
coupled with the luminescence oxygen channeling immunoassay (LOCI)
(E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426-5430,
1994). RMP and PZA resistances are associated with multiple mutations
within the rpoB and pncA genes, respectively.
M. tuberculosis genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP
resistance in a "blind study." Similarly, PZA resistance was
determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility
determination directly from cells of 10 clinical isolates grown in
culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing
result. Sequence analysis of the rpoB gene of this clinical
isolate revealed a single base substitution, most likely a silent point
mutation. The new BMI-LOCI mutation detection method is a rapid and
accurate procedure for the genotypic determination of the RMP and PZA
susceptibilities of M. tuberculosis clinical isolates. BMI
can also be detected by using commercially available automated
enzyme-linked immunosorbent assay plate formats (Lishanski et al.,
Nucleic Acids Res. 28:E42, 2000).
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INTRODUCTION |
The incidence of tuberculosis is
increasing in many countries, and control of the disease is threatened
by the emergence of drug resistance. In the past few years considerable
progress has been made in understanding the mechanisms of action of
antimycobacterial agents and the genetic basis of resistance to some of
these compounds (13). To date, there is information about 12 genes involved in resistance in Mycobacterium tuberculosis
(2, 14). There is a need for rapid susceptibility tests for
M. tuberculosis, particularly for first-line drugs such as
rifampin (RMP), pyrazinamide (PZA), isoniazid, and ethambutol. A test
method that is based on detection of mutations in the respective gene
sequences is likely to be a useful adjunct to the lengthy conventional
culture-based susceptibility testing and should prove useful for
improving the treatment and control of the disease.
The molecular basis of M. tuberculosis resistance to RMP has
been shown to be associated with deletion, insertion, or missense mutations in the 81-bp region of the rpoB gene, which
encodes the 
-subunit of the DNA-dependent RNA polymerase
(8). More than 96% of M. tuberculosis isolates
showing resistance to RMP have been reported to have one or two
mutations within this region (10). Thus, the detection of a
mutation in the rpoB gene can be used as an accurate marker
for detection of RMP resistance in the majority of M. tuberculosis strains tested. Furthermore, RMP resistance is
associated with multidrug resistance (3).
Resistance to PZA has been shown to be accompanied by loss of
pyrazinamidase (PZase) activity in M. tuberculosis
(10). PZase converts PZA to bactericidal pyrazinoic acid,
and loss of PZase activity is associated with PZA resistance. Multiple
mutations, which span the entire coding region of the pncA
gene, have been shown to be associated with PZA resistance in M. tuberculosis. Mutations in the pncA gene have been
identified as the cause for acquired PZA resistance in M. tuberculosis, and sequence alteration of this gene has also been
reported for the naturally resistant Mycobacterium bovis
strain (11, 12). The percentage of PZA-resistant M. tuberculosis strains that have alterations in the pncA
gene has been reported as between 72 and 97% (1). In order
to detect PZA-resistant strains, in vitro testing of the susceptibility of M. tuberculosis to PZA is highly recommended.
Unfortunately, conventional agar-based testing for PZA susceptibility
often leads to noninterpretable results because of insufficient growth
in the acidified medium (15).
Insofar as multiple mutations in the rpoB and
pncA genes have been shown to be associated with RMP and PZA
resistance, respectively, molecular methods for the genotypic
determination of resistance to these antibiotics should be designed to
detect the presence of any and all mutations in these genes. Several
methods for the detection of multiple mutations have been described in
recent years. Most of these methods require gel separation and are not equally effective for the detection of all sequence alterations. We
have recently described a method for rapid detection of any alteration
in a test nucleic acid sequence relative to a reference sequence, which
can be amplified by PCR (6).
The method is based on the detection of inhibition of branch migration
in four-stranded cruciform structures. The cruciform structures are
formed by annealing of partial duplexes prepared by amplification of
test and reference nucleic acid sequences using specially designed PCR
primers. If test and reference sequences are identical, the cruciform
structures resolve into undetectable duplexes; if a mutation is
present, the cruciform structures are stable and can be detected. This
novel mutation detection method, termed branch migration inhibition
(BMI), has been shown to be equally effective in the detection of any
mutation, including single-base substitutions, insertions, and
deletions. The method is particularly suitable for the genotypic
determination of drug susceptibility where the resistance phenotype is
associated with multiple mutations in a known genetic locus.
We describe the development and performance of a rapid assay for
genotypic determination of RMP and PZA susceptibilities of M. tuberculosis, employing the BMI mutation detection method
coupled with a homogeneous, light-induced luminescence detection
method, the luminescence oxygen channeling immunoassay (LOCI)
(16). The correct determination of drug susceptibility using
either purified genomic DNA or whole bacterial cells from
clinical specimens is demonstrated. The new method is simple, fast, and
amenable to automation.
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MATERIALS AND METHODS |
Clinical isolates.
Ten samples of M. tuberculosis
cells from clinical isolates were used in the study. The isolates, of
known RMP susceptibility phenotypes, grown either on Lowenstein-Jensen
agar or in Middlebrook 12B broth, were collected following different
growth periods, spun down, and resuspended in assay buffer. Cells were
harvested and heat killed prior to analysis. Three separate cell
disruption methods, sonication, microwave irradiation, and heating to
98°C for 10 to 15 min, were evaluated for feasibility for direct
genotypic determination of antibiotic resistance.
Purified genomic DNA samples.
A total of 64 genomic DNA
samples purified from M. tuberculosis clinical isolates were
used (17). These included 23 RMP-resistant isolates, 8 PZA-resistant isolates, and 33 wild-type isolates. Forty-six of these
genomic DNA samples (18 RMP-resistant isolates and 28 wild-type
isolates) were used in the blind study for the genotyping of RMP
resistance. Thirty-seven of the genomic DNA samples (8 phenotypically
PZA-resistant isolates and 29 wild-type isolates which were used in the
determination of RMP resistance) were used for the genotyping of PZA
resistance. Genomic DNA samples purified from four
Mycobacterium other than tuberculosis (MOTT) isolates, including Mycobacterium smegmatis,
Mycobacterium kansasii, M. bovis, and
Mycobacterium intracellulare, were also studied. Phenotypic
determination of the antibiotic susceptibilities of the isolates was
carried out at a reference laboratory (Northern California State
Reference Laboratory at Berkeley). RMP susceptibility testing was
carried out by agar proportion methods. PZA susceptibility testing was
performed by the radiometric method in 7H12 broth (2a).
M. tuberculosis genomic DNA or cells from clinical isolates were provided by the Stanford Medical Center (Palo Alto, Calif.). Purified human genomic DNA was obtained from the Coriell Institute for
Medical Research (Camden, N.J.).
DNA target sequences.
DNA target sequences were the 3,853-bp
M. tuberculosis rpoB gene (GenBank accession no. U12565),
the 561-bp M. tuberculosis pncA gene (GenBank accession no.
U59967), and the human cystic fibrosis gene (CFTR), exon 11.
Oligonucleotides.
All oligonucleotides were synthesized with
specific modifications by Oligos, Etc. (Wilsonville, Oreg.). The
sequences of the oligonucleotide primers used for mutation analysis are
listed in Table 1. All primers were 3'
modified by the addition of two ethenoadenosine residues. The added
nonhybridizing modified nucleotide, ethenoadenosine, was found to
provide added priming specificity when used with cloned Pfu
polymerase, which was from Stratagene (San Diego, Calif.). More recent
results of BMI with other DNA targets strongly suggest that the use of
ethenoadenosine primers is probably not necessary as long as
"hot-start" protocols are used (7).
DNA amplification.
PCR amplification of all test targets was
carried out using a 5'-biotin-labeled forward primer and two-tailed
reverse primers with "tail" sequences (which are not complementary
to the target) as shown in Fig. 1. The
reference target (wild-type M. tuberculosis) and nonrelevant
target (human CFTR exon 11) were amplified using the corresponding
5'-digoxigenin-labeled forward primer and two-tailed reverse primers
composed of a 3' target-specific portion and 5' tails which are the
same as those used for the test M. tuberculosis rpoB target
sequence.
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FIG. 1.
Generation of BMI-specific products. The formation of
four-stranded DNA structures and their resolution depending on the
presence of a mutation are described in the text. 5'-end-biotin (B)- or
digoxigenin (D)-labeled oligonucleotides are used as the forward (f)
primers for mutant target and wild-type reference sequences. The
"tails" in the reverse (r) primers are designated t1 and t2. The
BMI method is based on the detection of inhibition of branch migration
in four-stranded cruciform structures formed by annealing of partial
duplexes prepared by amplification of test and reference nucleic acid
sequences. The stable cruciform structures are presented within braces;
the rest are the unstable cruciform structures which will resolve and
separate, as well as the partial duplexes.
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A master reaction mixture (mix 1; 25 µl) containing 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, 1.5 mM MgCl
2, 0.2 mg of bovine serum
albumin (BSA)/ml, 200 µM each of the four deoxynucleoside
triphosphates,
and 250 nM each of the primers was aliquoted to each
reaction
tube containing a wax gem (Perkin-Elmer) to form the wax
barrier
on top of the liquid reaction mixture. A second reaction
mixture
(mix 2; 20 µl) containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl,
1.5 mM MgCl
2, 0.2 mg of BSA/ml, and 2.5 U of
Pfu
DNA polymerase/25
µl was also prepared and aliquoted to each reaction
tube following
formation of the wax barrier. Five microliters of the
test or
reference target was added to each tube, prepared as above. PCR
amplification was carried out in a T3-Thermoblock thermocycler
(Biometra Inc., Tampa, Fla.). Amplification of the
M. tuberculosis rpoB gene target sequence was carried out using the
following
thermocycling profile: 4 min at 95°C for denaturation of
target
DNA, followed by 39 cycles of 45 s at 95°C and 2 min at
70°C.
Amplification of the
M. tuberculosis pncA gene was
carried out
using the following thermocycling profile: 4 min at 95°C,
followed
by 39 cycles of 30 s at 95°C, 1 min at 64°C, and 1 min at 72°C.
Amplification of the human CFTR exon 11 gene sequence
was carried
out using the following thermocycling conditions: 4 min at
95°C,
followed by 39 cycles of 30 s at 95°C, 1 min at 64°C,
and 1 min
at 72°C.
BMI mutation analysis and signal detection.
BMI analysis of
test DNA amplification products was carried out by mixing reaction
mixtures of test samples with amplification products of either
relevant, wild-type M. tuberculosis reference genomic DNA or
a nonrelevant, human cystic fibrosis exon 11 gene reference sequence.
One microliter each of reference (wild-type) amplification products and
test amplification products were added to PCR tubes containing 4 µl
of 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, and 2 mg of BSA/ml. The mixtures were subjected to denaturation and
reannealing (2 min at 95°C followed by 30 min at 65°C). The
formation of stable cruciforms, indicating the presence of an
alteration(s) in the test sequence, was measured by LOCI, a homogeneous
singlet oxygen chemiluminescence immunoassay (16). Fifty
microliters of a LOCI bead suspension, containing 2.5 µg of
streptavidin-coated sensitizer particles and 1.25 µg of
anti-digoxigenin monoclonal antibody-coated chemiluminescer particles
was added, and the tubes were incubated at 37°C for 30 min to allow
binding of the particles. Chemiluminescence signals were read (3 cycles
of 1 s of illumination (680 nm) and 1 s of read [560 to 630 nm]) with an in-house-built reader. BMI analysis using the nonrelevant
reference was carried out similarly with the exception that the
postamplification reaction mixture of CFTR genomic DNA was first
diluted 10-fold, and 1 µl of this diluted reaction mixture was mixed
with 1 µl of the post-PCR mixture of the test sample.
Normalization of BMI signals.
A normalized BMI signal, as
related to the presence of sequence alterations, is obtained by
calculating the ratio of the signal generated from a mixture of the
test amplification product with the relevant reference amplification
product (the M. tuberculosis rpoB [GenBank accession no.
U12565] or pncA [U59967] gene) to the signal generated
from a mixture of the test amplification product with a nonrelevant
reference amplification product (the CFTR gene, exon 11 [cystic
fibrosis mutation database available at
http: //www.genet.sickkids.on.ca/cftr/]).
| |
RESULTS |
Experimental design.
The BMI mutation analysis method was
applied to the detection of sequence alterations in M. tuberculosis. The method is based on the generation from
related test and reference nucleic acid sequences of
amplification products capable of forming partial duplexes, which are
able to anneal to form four-stranded cruciform structures. When the
test and reference sequences are identical, spontaneous branch
migration occurs, and the cruciform structures formed by the
association of partial duplexes dissociate to full duplexes. However,
any sequence alteration in the test sequence relative to the reference
sequence will result in inhibition of branch migration and the
formation of stable cruciform structures (9). When suitable
labels are attached to the PCR primers, the cruciform structures can be
detected by various immunochemical methods.
We detect the stable cruciform structures by LOCI (
16) as
shown in Fig.
2. The binding of
sensitizer and chemiluminescer
particles to the respective labels of
the stable cruciform structures
results in the formation of complexes
capable of producing light-induced
chemiluminescence. Due to the short
lifetime of singlet oxygen
(about 4 µs), only chemiluminescer dyed
particles that are closely
associated with the sensitizer particles
will react to produce
chemiluminescence signals. Thus, particles that
are not attached
to the stable cruciform structures contribute only
minimally to
signal generation.
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FIG. 2.
LOCI detection of BMI products. The LOCI detection
method employs two different dyed latex particles, a streptavidin
(SAv)-labeled photosensitizer latex particle and an anti-digoxigenin
monoclonal antibody (anti-Dig MAb)-coated chemiluminescer latex
particle, as mentioned in the text. Illumination of the reaction
mixture with a light source of defined wavelength (680 nm) results in
production of singlet oxygen by the sensitizer dye, which can react
with the acceptor dye to produce chemiluminescence signals (550 nm).
Due to the short lifetime of singlet oxygen (4 µs), only
chemiluminescent particles that are closely associated with the
sensitizer particles will react to produce chemiluminescence signals.
The binding of sensitizer and chemiluminescer particles to the
respective labels of the cruciform structures results in the formation
of complexes capable of producing light-induced chemiluminescence. WT,
wild type; hv, laser light, with a defined wavelength.
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BMI analysis for the detection of rpoB mutations
associated with RMP resistance.
The performance of BMI for
detection of sequence alteration in test DNA compared to a reference
DNA is dependent on the ratio of signals obtained for the mutant and
wild-type genotypes. Optimization of conditions for formation of
cruciform structures and resolution of the structures when the test
sequence is of a wild-type genotype revealed dependency on both the
temperature and the Mg2+ ion concentration. Optimal
performance was achieved at 65°C for 30 min and was the same for the
various target systems tested. The detection of mutant M. tuberculosis genotypes was found to be optimal at 15 mM
Mg2. Analysis of genomic DNA samples purified from 10 M. tuberculosis clinical isolates revealed clear
discrimination of wild-type (n = 5) and mutant
(n = 5) rpoB genotypes, with mean BMI
signals for mutant genotypes (2,985,000 ± 156,000; coefficient of
variation [CV], 13%) about 20-fold higher than the mean signals for
the wild-type genotype (120,000 ± 17,000; CV, 5%).
Low BMI signals for mutant genotypes may result from inefficient
amplification or low initial sample DNA input and could be
incorrectly
interpreted as wild-type genotypes. We have developed
a method for
normalization of BMI signals, which corrects for
variations in input
test DNA or in amplification efficiency and
ensures correct genotype
determination. Partial duplexes generated
from the test and nonrelevant
reference DNA amplification products
can bind to each other by
hybridization of the complementary tail
sequences to form cruciform
structures. Because the sequences
of the double-stranded portions of
the partial duplexes are not
related, spontaneous branch migration does
not occur and the four-stranded
cruciform structures are
stable. Signals generated from these
structures are proportional
to the amount of test DNA in the sample
and are used for normalization
of the mutation-related
signals.
Genotypic determination of RMP resistance directly from cells.
The performance of BMI coupled with LOCI for detection of mutations in
the rpoB gene sequence directly from cells, i.e., without prior DNA purification, was assessed. The three cell disruption methods
were found to be equally efficient in terms of releasing DNA from
mycobacterial cells (data not shown).
Genotypic determination of RMP susceptibility directly from
heat-treated cells of 10
M. tuberculosis isolates was
carried
out using BMI analysis coupled with the LOCI detection method.
As shown in Table
2, the genotypic
determination of 9 out of
the 10 isolates correlated well with
phenotypic determination
of RMP susceptibility. Only one discrepancy
between the genotypic
determination and the conventional culture-based
phenotypic determination
of antibiotic resistance was observed.
Sequence analysis of the
rpoB gene of the discrepant
M. tuberculosis clinical isolate (isolate
8) revealed the
presence of a mutation in a phenotypically susceptible
isolate. Nucleic
acid sequencing of fragments of the
rpoB gene,
which include
the region of interest for genotypic determination
of RMP resistance,
was performed at the sequencing facility of
Stanford University.
Sequence analysis was obtained for isolate
8, a phenotypically
resistant strain (isolate 7) derived from
it, and a wild-type isolate.
The sequence of the fragment derived
from the wild-type strain was
identical to that reported by Telenti
(
14). A point mutation
at position 198 (according to the numbering
of Telenti), with a
replacement of C by T, was revealed for the
isolate for which there is
a discrepancy between the genotypic
and phenotypic determinations. This
new point mutation is most
likely a silent mutation, which does not
alter the function of
the RNA polymerase. Other silent mutations in
rpoB have been described
previously (
4). Sequence
information for the
rpoB fragment
of the phenotypically
resistant strain derived from the above
isolate revealed a point
mutation at position 198, as well as
an additional replacement of C by
T at position 248. The point
mutation at position 248 was previously
described as conferring
RMP resistance.
Blind study of genotypic determination of RMP resistance.
A
panel of 46 genomic DNA samples was tested in a "blinded" fashion
for rpoB genotype. In addition to BMI analysis of the test
gene relative to a wild-type reference gene, a method for the
normalization of BMI signals was also employed as described above.
Amplification of a CFTR sequence, exon 11, was carried out using the
primer oligonucleotides specified in Table 1, and the product was
employed as a nonrelevant reference nucleic acid sequence for the
normalization procedure.
The results obtained by using this normalization scheme are summarized
in Fig.
3. The normalized BMI-LOCI signal
clearly differentiates
between mutant and wild-type isolates. The
mutant and wild-type
genotype determinations by this method are in full
agreement with
the phenotypic results for the clinical isolates as
determined
by conventional culture-based methods at the Stanford
Medical
Center.
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FIG. 3.
M. tuberculosis rpoB genotypic determination:
blind study. Shown are normalized BMI signals for analysis of
rpoB gene mutations. A total of 46 M. tuberculosis genomic DNA samples were analyzed by PCR-BMI-LOCI
using a wild-type M. tuberculosis amplicon as the relevant
reference and CFTR exon 11 as the nonrelevant reference to normalize
signals. Samples 1 to 28 represent RMP-susceptible isolates, and
samples 31 to 48 are RMP resistant. A normalized BMI signal is obtained
by calculating the ratio of the signal generated from a mixture of the
test amplification product with the relevant reference amplification
product (S1) to the signal generated from a mixture of the test
amplification product with the nonrelevant reference amplification
product (S2).
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Genotypic determination of PZA resistance.
Oligonucleotide
primers (Table 1) were selected for efficient amplification of the
pncA gene, followed by BMI mutation analysis coupled with
LOCI detection for the genotypic determination of PZA susceptibility.
Amplification of the pncA gene sequence results in the
generation of a 620-bp amplicon. Thirty-seven DNA samples purified from
M. tuberculosis clinical isolates were tested for genotypic
determination of PZA susceptibility. These included 29 phenotypically
wild-type isolates (used in the determination of RMP resistance) and 8 phenotypically PZA-resistant isolates. The results of genotypic
determination of PZA susceptibility are shown in Fig.
4, demonstrating perfect correlation of
the phenotypic and genotypic determinations, with very strong
discrimination between susceptible and resistant genotypes.
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FIG. 4.
M. tuberculosis pncA gene mutation detection
by PCR-BMI-LOCI. A total of 37 genomic DNA samples (samples 1 to 29 are
wild-type isolates, and samples 31 to 38 are PZA-resistant isolates)
were analyzed using BMI-specific primer sets. The amplification product
of wild-type M. tuberculosis was used as a reference. The
amplicon size is 620 bp.
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Specificity of the BMI genotypic determination of antibiotic
resistance.
The specificity of the BMI-LOCI method for detection
of M. tuberculosis mutant RMP and PZA genotypes was
examined. In the event that the clinical isolates contain an additional
mycobacterial species, it is possible that any sequence difference
between the reference (M. tuberculosis wild-type sequence)
and the nontuberculosis sequence will result in the generation of a
positive BMI-LOCI signal, which may be misinterpreted as indicative of
a resistant M. tuberculosis genotype. Thus, we have studied
the BMI-LOCI responses obtained from genomic DNAs of a number of
mycobacterial species. This study was undertaken only for the
validation of the BMI-LOCI method for determination of M. tuberculosis antibiotic resistance. Genomic DNAs purified from
four MOTT isolates, including isolates of M. smegmatis, M. kansasii, M. bovis, and
M. intracellulare, were used in this study and tested
together with wild-type and mutant genomic DNA samples of M. tuberculosis. All but M. bovis yielded low BMI signals,
similar to signals obtained from wild-type M. tuberculosis
DNA, as shown in Fig. 5 and
6. The BMI-LOCI analysis of
M. bovis rpoB DNA showed a wild-type genotype,
whereas analysis of the pncA gene sequence showed a mutant
genotype. This result is in accordance with known sequence alterations
resulting in the naturally PZA-resistant phenotype of M. bovis.
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FIG. 5.
Species specificity of detection of mutations in the
M. tuberculosis rpoB gene. BMI analysis was carried out
using two PCR primer sets. The same forward primer (1779 5'-biotin or
5'-digoxigenin) was used with two sets of reverse tail primers (1963t1
and 1963t2; 2044t1 and 2044t2) with amplicon sizes of 225 and 306 bp,
respectively. Samples included genomic DNA purified from two wild-type
M. tuberculosis isolates (wt1 and wt2), two RMP-resistant
M. tuberculosis isolates (mut1 and mut2), and four MOTT
isolates (M. smegmatis, M. kansasii, M. bovis, and M. intracellulare). An amplification product
from wild-type M. tuberculosis was used as a reference.
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FIG. 6.
Species specificity of detection of mutations in the
M. tuberculosis pncA gene. BMI analysis was carried out
using a PCR primer set which generates 620-bp amplicons. Samples
included genomic DNA purified from five wild-type M. tuberculosis isolates (wt1 to wt5), five PZA-resistant M. tuberculosis isolates (mut1 to mut5), and four MOTT isolates
(M. smegmatis, M. kansasii, M. bovis,
and M. intracellulare). An amplification product from
wild-type M. tuberculosis was used as a reference.
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Nonspecific PCR amplification leads to increased background
signals and reduced signal-to-background ratios. Primer selection,
together with a hot-start PCR amplification procedure, was found
to be
essential for reducing background signals. The performances
of two
PCR-BMI primer pairs for analysis of
rpoB gene mutations
are
shown in Fig.
5. Although high signal-to-background (S/B)
ratios were
obtained with both primer pairs, this performance
parameter (S/B, 37 for the primer set of 1779 [forward]-1963t1
and -t2 [reverse]
versus 90 for the primer set of 1779 [forward]-2044t1
and -t2
[reverse]) can be improved with proper primer pair
selection.
Reproducibility of the BMI-LOCI signals for genotypic determination
of antibiotic resistance.
To assess assay reproducibility, eight
independent replicate determinations of two DNA samples, one from a
phenotypically wild-type and one from a phenotypically PZA-resistant
M. tuberculosis isolate, were carried out as described
before. Very reproducible results were obtained. The mean value of the
eight wild-type signals is 22,136 ± 1,528 (CV, 6.9%), and the
mean value of the eight mutant signals is 2,283,981 ± 106,972 (CV, 4.6%).
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DISCUSSION |
The results reported demonstrate the feasibility of the BMI-LOCI
mutation detection method for genotypic testing of resistance to two
antituberculosis agents, RMP and PZA. Multiple mutations in the
rpoB and pncA gene sequences have previously been
reported to be associated with RMP and PZA resistance (11,
13) and are present in the majority of resistant isolates.
Application of a rapid DNA-based assay would be of benefit for
determining drug susceptibility patterns (13). Detection of
RMP resistance should also identify multidrug-resistant strains, nearly
all of which are resistant to RMP (3).
Correct genotypic determination of PZA- and RMP-resistant M. tuberculosis using BMI analysis coupled with the LOCI detection method was demonstrated by employing genomic DNA purified from clinical
isolates. Mutations in the rpoB and pncA genes
were identified in all genomic DNA samples purified from RMP- and
PZA-resistant isolates tested. Further validation of the BMI method for
genotypic identification of RMP- and PZA-resistant M. tuberculosis will require testing of additional clinical isolates,
preferably selected from diverse geographic settings so as to ensure
that the full spectrum of resistance-conferring and silent mutations is sampled.
Nine out of 10 BMI genotypic determinations of RMP susceptibility
directly from cells correlated well with phenotypic determinations by a
culture-based method. Sequence analysis of the rpoB gene segment used for genotypic determination revealed a point mutation, which had not been previously described, in the single discrepant isolate. The new mutation is likely to be a silent mutation which gives
a positive BMI-LOCI signal in this phenotypically susceptible isolate.
Other silent rpoB mutations have been described previously (4).
The BMI method detects only the presence, not the type, of mutations. A
method like BMI that is independent of the type of mutation would be
very useful in antimicrobial susceptibility testing, when any of the
multiple mutations within a given sequence can often give rise to drug
resistance. Insofar as the new mutation detection method is capable of
detecting any mutation in the gene sequence tested, it is expected to
detect silent mutations as well as mutations leading to a mutant
phenotype. The potential for false classification of isolates with
mutant genotypes as antibiotic resistant is ever present when one is
using a nucleic-acid-based method which detects any sequence alteration
in the test sample relative to a reference. The practical impact of
this potential discrepancy between phenotypic and genotypic
determinations is yet to be determined. However, this disadvantage is
likely small compared to the benefits of rapid screening of clinical
M. tuberculosis isolates for drug resistance.
A simple method for the normalization of BMI-LOCI signals obtained from
various test samples was also developed. This method is useful for
overcoming the possible false genotypic determination of phenotypically
antibiotic-resistant isolates as susceptible. This false determination
could occur as a consequence of a low signal resulting from either a
low input of DNA or amplification failure. Both of these causes for
potential false-negative results are common for all nucleic-acid-based
methods and would have significant impact on the clinical utility of
the genotyping method. Another method for checking PCR success is to
add ethidium bromide to the PCR mixture (7). The ability to
control for these potential amplification failures will facilitate the
use of the new method as a rapid screen for drug-resistant M. tuberculosis.
In conclusion, the feasibility of rapid genotypic determination of
M. tuberculosis RMP and PZA resistance using the BMI
mutation detection method was demonstrated. Coupled with the LOCI
homogeneous detection method, the new procedure is rapid, highly
reproducible, and specific. Other detection methods, such as the
enzyme-linked immunosorbent assay, are also feasible, as was shown
previously (6). The ability to detect mutations in specific
nucleic acid sequences directly from mycobacterial cells, without prior
purification of genomic DNA, should render this method suitable for the
clinical microbiology laboratory.
| |
ACKNOWLEDGMENTS |
We acknowledge the contributions of A. Lishanski and E. Ullman
through helpful discussions and thank S. Rose and A. Dafforn for
reviewing this paper and M. Taylor for assistance in computer graphics.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Advanced
Diagnostics Division, Dade Behring Inc., 3403 Yerba Buena Rd. (M/S
E1-304), San Jose, CA 95135. Phone: (408) 239-2081. Fax: (408)
239-2707. E-mail: yenping_liu{at}dadebehring.com.
| |
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Journal of Clinical Microbiology, October 2000, p. 3656-3662, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
