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Previous Article | Next Article 
Journal of Clinical Microbiology, January 1998, p. 239-242, Vol. 36, No. 1
Servicio de Microbiología,
Received 12 May 1997/Returned for modification 2 September
1997/Accepted 21 October 1997
An allele-specific amplification method based on two genetic
polymorphisms to differentiate Mycobacterium tuberculosis
from Mycobacterium bovis was tested. Based on the
differences found at position 169 in the pncA genes from
M. tuberculosis and M. bovis, a PCR system
which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species
was developed. All 121 M. tuberculosis strains showed the
expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless,
18 of the 116 M. bovis isolates, all of them goat isolates,
showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the
M. tuberculosis species. Based on the polymorphism found at
position 285 in the oxyR gene, the same system was used to
differentiate M. tuberculosis from M. bovis. In
this case, DNAs from all 121 M. tuberculosis isolates had
the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the
identical polymorphism (adenine). The oxyR allele-specific
amplification method can differentiate M. bovis from
M. tuberculosis, is rapid (results can be obtained in less
than 3 h), and is easy to perform.
While most cases of human
tuberculosis are caused by Mycobacterium tuberculosis,
infections caused by Mycobacterium bovis are being
increasingly documented, in some cases in epidemic nosocomial bursts
(2, 3, 8). Classical methods to differentiate both species
are based on nitrate reduction, pyrazinamidase activity, pyrazinamide
susceptibility, niacin accumulation, and growth in thiophene-2-carboxylic acid hydrazide-containing media. Some of these
assays are tedious and time-consuming and are infrequently performed by
diagnostic laboratories. However, from the clinical and epidemiological
perspective, a rapid method to differentiate M. tuberculosis
from M. bovis is urgently needed, both for treatment (due to
the intrinsic resistance of M. bovis to pyrazinamide) and
eventually for attribution of the isolate to a previously known
epidemic outbreak.
The mtp40 gene, considered to be present in M. tuberculosis but absent in M. bovis, was isolated and
characterized several years ago (17). PCR amplification of
this gene was initially used to differentiate M. tuberculosis from M. bovis (4).
Nevertheless, this method has recently been invalidated since the gene
has been documented as not present in all M. tuberculosis
strains and not absent in all M. bovis strains
(25). The nucleotide sequence of the katG gene
from M. bovis is identical to that of M. tuberculosis except that M. bovis contains a thymine
instead of a guanine at position 1388 (21). This
polymorphism is not reliable to differentiate among both species since
there are some strains of M. tuberculosis containing the
cited change. In 1996, the pncA gene, a gene involved in
pyrazinamide sensitivity, was described (20). Strains
containing specific mutations in this gene showed increased resistance
to pyrazinamide. Interestingly, all M. bovis strains tested
had an identical mutation in the nucleotide sequence of the gene (G
instead of C at nucleotide position 169). This change caused a
substitution of histidine for aspartic acid at position 57, which
results in an inactive pyrazinamidase enzyme. These authors found that
all investigated M. bovis strains showed the same change.
Finally, Sreevatsan et al. (21, 22) identified another
polymorphic nucleotide specific for M. bovis in the
oxyR gene. All M. bovis strains sequenced had an
adenine residue at nucleotide 285, whereas all M. tuberculosis strains had a guanine residue at this position. Therefore, polymorphisms in pncA and oxyR are
good candidates to develop a method to rapidly differentiate M. bovis from M. tuberculosis. Based on the
pncA polymorphism, Scorpio et al. developed a single-strand
conformational polymorphism assay to distinguish M. tuberculosis from M. bovis (18). This method
is rapid but requires the use of acrylamide gels (after verification of
the success in amplification). Based on the oxyR
polymorphism, Sreevatsan et al. (21) developed a
PCR-restriction fragment length polymorphism (PCR-RFLP) method. In this
case, a 548-bp fragment of oxyR is amplified. Agarose gel
electrophoresis is performed to verify the amplification of the desired
fragment before AluI digestion. The result of the
restriction is visualized by a new electrophoresis.
In the last few years, several methods that detect single nucleotide
changes in DNA fragments have been described (for a review, see
reference 6). The allele-specific PCR method was
originally described in 1989 (14) and later improved
(7, 13). In this report, we describe the use of an
allele-specific PCR method to detect the polymorphisms in the
pncA and oxyR genes with the objective of rapidly
and easily differentiating M. bovis from M. tuberculosis. The first system we tested was based on the
nucleotide polymorphism at position 169 in the pncA gene.
This system failed to differentiate M. bovis isolated from
goats from M. tuberculosis strains. To overcome this
limitation, we assayed another polymorphism, the guanine or adenine at
position 285 in the oxyR gene. The diagnostic possibilities
of both systems were evaluated with 237 strains belonging to the
M. tuberculosis complex.
(This work was presented in part at the 37th Interscience Conference on
Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997.)
Strains and DNA.
The M. tuberculosis strains used
in this study were either isolated from different patients from the
Hospital Infantil de México Federico Gómez, México
D.F., México (80 strains), or the Hospital Ramón y Cajal,
Madrid, Spain (31 strains), or were from the Universidad de Zaragoza
collection (10 strains). The M. bovis isolates were from the
Facultad de Medicina, Universidad de Zaragoza (105 strains), the
Facultad de Veterinaria of the Universidad Complutense de Madrid (10 strains), and the Hospital Ramón y Cajal (1 strain). The M. tuberculosis strains were isolated from human patients. The
M. bovis strains had different origins, including humans,
cattle, and goats. Chromosomal DNA was purified as previously described
(16).
Species characterization.
M. tuberculosis complex
determination was made by classical methods (12) and by the
PCR restriction enzyme pattern analysis method previously described
(24). This method is based on the different restriction
enzyme patterns of a PCR-amplified DNA fragment in the gene encoding
the 65-kDa mycobacterial protein. Previously described biochemical
tests were used to distinguish M. bovis from M. tuberculosis (10, 12, 15).
Primers and PCR conditions.
The amplification primers for
pncA and oxyR used in this study were based on
previously described sequences (5, 20). For the
allele-specific pncA method, the forward primer, pncATB-1.2 (5'-ATGCGGGCGTTGATCATCGTC-3'), complements bases 1 to 21 in
the gene. The reverse primers, pncAMT-2
(5'-CGGTGTGCCGGAGAAGCGG-3') and
pncAMB-2 (5'-CGGTGTGCCGGAGAAGCCG-3'),
complement bases 185 to 168. For the oxyR
allele-specific method, the forward primer was oxyRTB-2.1
(5'-TGGCCGGGCTTCGCGCGT-3') and the reverse primers were
oxyRMT-1 (5'-GCACGACGGTGGCCAGGCA-3')
and oxyRMB-1 (5'-TGCACGACGGTGGCCAGGTA-3'). (The
underlined bases are changes included in the primer sequences and do
not complement the published sequences. The boldfaced bases complement
those present in the M. tuberculosis or M. bovis
sequences.) For each allele-specific method, DNA samples were subjected
to two differential amplifications in two separate tubes. Both
reactions were performed with the same forward primer, pncATB-1.2 or
oxyRTB-2.1, and one of the two discriminator primers, pncAMT-2 or
oxyRMT-1 for M. tuberculosis or pncAMB-2 or oxyRMB-1 for
M. bovis. The M. tuberculosis-specific amplification reaction mixture contained 1 µl of template DNA (about
100 ng) and 24 µl of a solution consisting of the following: 200 µM
each deoxynucleoside triphosphate, 25 pmol of each primer (pncATB-1.2
and pncAMT-2 for pncA or oxyRTB-2.1 and oxyRMT-1 for oxyR), 1.0 U of Taq-Gold DNA polymerase
(Perkin-Elmer), 10 mM Tris hydrochloride (pH 8.3), 50 mM KCl, 2 mM
Mg2Cl, and 0.01% gelatin. The M. bovis-specific
amplification reaction mixtures were identical to those of M. tuberculosis but contained primer pncAMB-2 instead of pncAMT-2,
for pncA, and oxyRMB-1 instead of oxyRMT-1, for
oxyR. For the pncA method, the cycling parameters were 95°C for 12 min, followed by 30 three-step cycles including denaturation at 94°C for 1 min, annealing at 67°C for 1 min, and extension at 72°C for 1 min. For the oxyR method, the
cycling parameters were 95°C for 12 min, followed by 30 two-step
cycles including denaturation at 94°C for 45 s and annealing
plus extension at 70°C for 1 min 30 s. The amplification
products were analyzed by electrophoresis on a 2% agarose gel and
visualized by ethidium bromide fluorescence. A unique amplification
product of 185 bp (pncA) or 270 bp (oxyR) must be
visualized in either M. tuberculosis or M. bovis
reactions.
Design of allele-specific primers and optimization of PCR
conditions.
A common forward primer, pncATB-1.2, and two reverse
allele-specific primers, pncAMT-2 and pncAMB-2, were designed to
hybridize with the published sequences of M. tuberculosis
and M. bovis pncA genes. The reverse primer
pncAMT-2, which was designed to anneal only to the
pncA sequence from M. tuberculosis and not to
that from M. bovis, has a mismatch in the third nucleotide
from the 3' end (corresponding to position 170 in the gene). It is
expected that this mismatch will prevent amplification only when the
template sequence also contains an additional change. The M. tuberculosis pncA gene complements pncAMT-2 except for the
mismatch, while the M. bovis gene, which has a change from C
to G at position 169, shows two mismatches, one at position 170 and the
other at position 169, corresponding to the polymorphism that
differentiates M. tuberculosis from M. bovis. In
contrast, the amplification of M. bovis pncA, but not that
of M. tuberculosis, will occur when the reverse primer for
M. bovis, pncAMB-2, is used. Figure 1 shows the principle of the method.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Allele-Specific PCR Method Based on pncA
and oxyR Sequences for Distinguishing Mycobacterium
bovis from Mycobacterium tuberculosis:
Intraspecific M. bovis pncA Sequence
Polymorphism
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (26K):
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FIG. 1.
Diagram showing the principle of the allele-specific PCR
method to detect the polymorphism at position 169 in the
pncA gene. pncATB-1.2 is a forward primer able to hybridize
both M. tuberculosis and M. bovis pncA genes;
pncAMT-2 is a reverse primer presenting a single mismatch with the
M. tuberculosis pncA gene and two mismatches with the
M. bovis gene; pncAMB-2 is a reverse primer presenting two
mismatches with the M. tuberculosis pncA gene and a single
mismatch with the M. bovis gene. Only single mismatches
allow PCR amplification. The identical principle was applied to detect
oxyR polymorphism.
Rapid differentiation of M. bovis from M. tuberculosis by allele-specific PCR. Different results were obtained with the pncA and oxyR systems.
(i) pncA system. The pncA allele-specific PCR system was applied to the control strain M. tuberculosis H37Rv (ATCC 27294) and 100 M. tuberculosis strains of human origin (México D.F., Zaragoza, and Madrid, Spain). In all cases, a positive amplification was obtained with the M. tuberculosis-specific primer pncAMT-2 but not with the M. bovis-specific primer pncAMB-2.
In M. tuberculosis strains, the acquisition of mutations rendering the product of pncA inactive is considered to result in pyrazinamide resistance. The change at position 169 of the gene from M. bovis is responsible for the lack of pyrazinamidase activity (which explains its natural resistance to the compound). The existence of pyrazinamide-resistant M. tuberculosis strains having this mutation cannot be ruled out. To test this possibility, we performed the pncA allele-specific amplification reactions with DNA extracted from 21 pyrazinamide-resistant M. tuberculosis strains of different origins. In all cases, a positive amplification fragment of 185 bp was obtained with the M. tuberculosis-specific reverse primer but not with the M. bovis one. This indicates that none of these strains contains a mutation at position 169. The same protocol was applied to 116 M. bovis strains of animal origin (Zaragoza and Madrid) and to 1 M. bovis strain of human origin (Madrid). With 99 of 117 (85%) of these strains, a positive amplification was obtained with pncAMB-2, and M. bovis-specific primer, but not with pncAMT-2. The existence of 18 strains of M. bovis not containing the expected M. bovis pncA polymorphism casts doubt on the validity of this system for use as a discriminating technique. Interestingly, the 18 strains were, in all cases, isolated from goats. Figure 2 shows the results of the pncA allele-specific amplification performed with DNAs from representative strains of bovine M. bovis, goat M. bovis, and M. tuberculosis. The entire nucleic acid sequences of the pncA genes from six M. bovis strains (three isolated from goats, two from cattle, and one from a human) were obtained. pncA genes from three M. tuberculosis strains were also sequenced. The sequences obtained fit the described polymorphisms (data not shown).
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(ii) oxyR system. In contrast to the results with the pncA system, all of the M. bovis strains tested (including those isolated from goats) had the described M. bovis-specific polymorphism (an adenine residue at position 285) in the oxyR gene. In addition, all M. tuberculosis strains had a guanine residue at the cited position. Figure 2 shows the results of the oxyR allele-specific amplification performed with DNAs from representative bovine and goat M. bovis and M. tuberculosis strains.
Sensitivity of the technique. To determine the sensitivity of the technique, both M. tuberculosis and M. bovis DNAs were serially diluted and subjected to amplification by the two cited systems. Amplicons of 185 or 274 bp were visualized when amplifications were performed with as little as 20 pg of chromosomal DNA. Both systems, pncA and oxyR, had similar sensitivities (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
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DNA sequencing has shown that pncA (20) and oxyR (5) genes from M. bovis strains contain changes at positions 169 and 285, respectively, with respect to the M. tuberculosis sequence. Based on these polymorphisms, single-strand conformational polymorphism (18) and PCR-RFLP (21) assays were developed. Our allele-specific PCR tests applied the same principle as that of these previous assays but with the advantages of having a shorter response time (less than 3 h) and being simpler and safer (no acrylamide gels required) as well as inexpensive (minimal volume of reagents and no restriction enzymes). Our results with oxyR confirm those of Sreevatsan et al. (21, 22), who found that the polymorphic site located at nucleotide 285 uniformly distinguished M. bovis (adenine) from non-M. bovis isolates. Furthermore, our work shows that the allele-specific PCR based on the oxyR polymorphism at nucleotide 285 is a useful technique for distinguishing M. bovis from M. tuberculosis.
The study of polymorphism at nucleotide 169 in the pncA gene shows that not all M. bovis strains contain the same nucleotide (guanine) at this position. In a recent study, Sreevatsan et al. (23) found a cytosine instead of a guanine at position 169 in two M. bovis isolates. Interestingly, all 18 M. bovis strains in our study presenting this particular substitution were isolated from goats. The position-169 polymorphism test was also performed with a large number of M. tuberculosis strains (131 strains), including 21 pyrazinamide-resistant strains. In all cases, the absence of the C-to-G mutation at position 169 was documented. This observation also confirms those made by Sreevatsan et al. (23) and Scorpio et al. (19), who did not find the M. bovis pncA-specific mutation in pyrazinamide-resistant M. tuberculosis strains.
By introducing the functional M. tuberculosis pncA gene into M. bovis BCG strains, Scorpio and Zhang (20) restored the pyrazinamide sensitivity of these strains. With this experiment, they demonstrated that the His-to-Asp change at position 57 in the pncA product is actually the change responsible for the lack of pyrazinamidase and nicotinamidase activities. The absence of this change among the almost 100 pyrazinamide-resistant isolates previously tested (21 from this study) (19, 23) shows that this mutation is not present, or very uncommon, among pyrazinamide-resistant M. tuberculosis strains. The explanations for the natural occurrence of a C-to-G mutation at position 169 in M. bovis remain unclear, considering that the same mutation appears to be unfavorable for pyrazinamide-resistant M. tuberculosis.
Organisms containing genes that encode active enzymes may evolve (by mutation and selection) increased enzyme activity in environments where the resulting phenotype provides a higher fitness. A decrease or loss of the activity may also be the result of a reduced fitness associated with exposure of the phenotype to another environment. M. tuberculosis has been suggested to have arisen from M. bovis (11), but that implies an ancestral M. bovis with nicotinamidase activity. Molecular epidemiology studies have recently shown that M. bovis strains isolated from goats cluster in specific spoligotyping (spacer oligonucleotide typing) and RFLP groups, very different from those in other M. bovis and M. tuberculosis strains (1, 9). These M. bovis strains showed a mixed phenotype according to the classic biochemical characteristics differentiating M. bovis from M. tuberculosis and, in particular, have a nicotinamidase-pyrazinamidase activity (13a). Considering the results shown here, it is tempting to speculate that the immediate missing-link ancestor of M. tuberculosis, if indeed represented by an extant lineage, may be the goat M. bovis strains and not the cattle M. bovis strains, as was proposed previously (11).
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ACKNOWLEDGMENTS |
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The investigators from the Madrid groups were supported in part by the Consejería de Educación y Cultura de la Comunidad Autónoma de Madrid (grant 0702596). The investigators from the Zaragoza group were supported in part by the Spanish Fondo de Investigaciones Sanitarias de la Seguridad Social (grant FIS/0051) and by the European Commission CA on the Molecular Epidemiology and Control of Tuberculosis BIOMED2 (grant CT9396-2102). L. E. Espinosa de los Monteros was the recipient of a fellowship from the Patronato del Hospital Infantil de México Federico Gómez. J. C. Galán was the recipient of a fellowship from the Fondo de Investigaciones Sanitarias (FIS/97-5520).
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FOOTNOTES |
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* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Ramón y Cajal, Ctra. Colmenar km 9.100, 28034 Madrid, Spain. Phone: 34-1-336 83 30. Fax: 34-1-336 88 09. E-mail: jesus.blazquez{at}hrc.es.
Present address: Servicio de Microbiología, Hospital
Infantil de México Federico Gómez, México 06720 D.F., México.
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