Rapid differentiation of Mycobacterium bovis and
Mycobacterium tuberculosis based on a 12.7-kb
fragment by a single tube multiplex-PCR
C.S. Bakshia, D.H. Shaha, 1, , Rishendra Vermab,
,
, R.K. Singha and
Meenakshi Malika
aNational
Biotechnology Center, Indian Veterinary Research Institute, Izatnagar,
Uttar Pradesh 243122, India
bMycobacteria
Laboratory, Division of Biological Standardization, Indian Veterinary
Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India
Received 18 June 2004; revised 25 January 2005; accepted 20 May 2005.
Available online 6 July 2005.
Abstract
The aim of this work was the design and validation of a rapid and easy single tube
multiplex-PCR (m-PCR) assay for the unequivocal differential detection of
Mycobacterium bovis and Mycobacterium tuberculosis. Oligonucleotide primers
were based on the uninterrupted 229-bp sequence in the M. bovis genome and a
unique 12.7-kb insertion sequence from the M. tuberculosis genome, which is
responsible for species-specific genomic polymorphism between these two closely
related pathogens. The m-PCR assay was optimized and validated using 22 M.
bovis and 36 M. tuberculosis clinical strains isolated from diverse host species and
9 other non-tuberculous mycobacterial (NTM) strains. The designed primers
invariably amplified a unique 168-bp (M. bovis-specific) and 337-bp (M.
tuberculosis-specific) amplicon from M. bovis and M. tuberculosis strains,
respectively. The accuracy of the assay, in terms of specificity, was 100%, as none
of the NTM strains tested revealed any amplification product. As little as 20 pg of
genomic DNA could be detected, justifying the sensitivity of the method. The mPCR assay is an extremely useful, simple, reliable and rapid method for routine
differential identification of cultures of M. bovis and M. tuberculosis. This m-PCR
may be a valuable diagnostic tool in areas of endemicity, where bovine and human
tuberculosis coexist, and the distinction of M. bovis from M. tuberculosis is required
for monitoring the spread of M. bovis to humans.
Keywords: Mycobacterium tuberculosis; Mycobacterium bovis; PCR
Article Outline
1. Introduction
2. Materials and methods
2.1. Mycobacteria strains and DNA
2.2. Species characterization
2.3. Primers and PCR conditions
2.4. Specificity and sensitivity of multiplex PCR assay
3. Results
3.1. Design of primers and optimization of m-PCR conditions
3.2. Rapid differentiation of M. bovis and M. tuberculosis by m-PCR
3.3. Sensitivity and specificity of m-PCR
4. Discussion
Acknowledgements
References
1. Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most
widespread infectious diseases and the leading cause of death, due to a single
infectious agent, among adults in the world. Likewise, bovine TB, caused by
Mycobacterium bovis, is a significant veterinary disease that can spread to
humans. M. tuberculosis is the most common cause of human TB, but an unknown
proportion of cases occur due to M. bovis (Acha and Szyfres, 1987). Moreover,
disease caused by M. bovis in human immunodeficiency virus (HIV)-positive
individuals is also an increasing concern (Daborn et al., 1993 and Blazquez et al.,
1997). There is a direct correlation between M. bovis infection in cattle and disease
in the human population (Cosivi et al., 1998). In industrialized countries, animal TB
control and elimination programs, together with milk pasteurization, have drastically
reduced the incidence of disease caused by M. bovis in both cattle and humans. In
developing countries, however, animal TB is widely distributed, control measures
are not applied or are applied sporadically, and pasteurization is rarely practiced.
For instance, M. bovis infection is responsible for about 2% and 8% of new cases
of human pulmonary and extra-pulmonary TB, respectively, in Latin America
(Cosivi et al., 1998). In Asia, 94% and >99% of the total cattle and buffalo
populations, respectively, are found in countries where bovine TB is either partly
controlled or not controlled at all. Thus, 94% of the Asian human population lives
and is at a risk, in countries where cattle and buffaloes undergo no or only limited
control for bovine TB (Cosivi et al., 1998). M. bovis and M. tuberculosis are closely
related organisms and have almost identical genomes (Imaeda, 1985). Despite the
high degree of DNA homology between the two organisms, M. tuberculosis causes
disease almost exclusively in humans and rarely in other animals, whereas M.
bovis can cause TB in a wide range of animal hosts, including humans (WHO,
1994). In areas where bovine and human TB coexist and are endemic, the
separation of M. bovis from M. tuberculosis is important in monitoring the spread of
M. bovis to humans. In addition, such a distinction has an impact on the treatment
of the disease, since M. bovis strains are naturally resistant to pyrazinamide (PZA)
(Konno et al., 1967); thus, human TB caused by M. bovis cannot be treated with
PZA.
Currently, differentiation of M. bovis from M. tuberculosis is based on conventional
culture and biochemical tests. In addition to being tedious and slow, current
methods, such as biochemical typing, are not 100% reliable due to the advent of
intermediate strains, such as niacin and T2CH variant M. tuberculosis (Verma et
al., 1987 and Grange et al., 1996) and PZA variant M. bovis (Niemann et al.,
2000). Partly due to close genetic relatedness and variable biochemical patterns,
definitive detection of M. bovis and M. tuberculosis, up to species level, is time
consuming and difficult. Methods, such as PCR, could be the best alternative
strategy to meet this purpose.
Recently, sequence analysis of the M. tuberculosis and M. bovis genomes has
shown that M. bovis lacks a 12.7-kb fragment present in the genome of M.
tuberculosis (Zumarraga et al., 1999). Further analysis of the 12.7-kb fragment
suggested that it represents a deletion in M. bovis rather than an insertion in M.
tuberculosis. This deletion removes most of the mce-3 operon, one of the four
closely related operons, which may be involved in cell entry. Therefore, it was
suspected that this deletion might contribute to differences in virulence or host
range in the two species. Interestingly, all the M. tuberculosis isolates studied
showed the presence of the 12.7-kb fragment, while all the M. bovis strains lacked
this fragment (Zumarraga et al., 1999). Therefore, the 12.7-kb fragment may be a
useful marker to differentiate M. bovis from M. tuberculosis. In this report, we
describe a new single tube multiplex-PCR (m-PCR) assay, based on a 12.7-kb
fragment, for the rapid and easy differential detection of M. bovis and M.
tuberculosis.
2. Materials and methods
2.1. Mycobacteria strains and DNA
M. tuberculosis strains (35) used in this study were clinical strains, i.e. isolates from
human patients with pulmonary TB from the Medical Hospital, IVRI, Izatnagar (UP),
India (9 strains), District Tuberculosis Hospital, Bareilly (UP), India (15 strains),
bovines (9 strains), guinea pig (1 strain) and swine (1 strain). M. bovis strains (20)
were isolated from bovines (17 strains), deer (2 strains) and black buck (1 strain).
The standard strains examined were M. tuberculosis H37Rv, M. bovis AN5, M.
bovis BCG (Mycobacteria Laboratory, IVRI), M. paratuberculosis (Teps strains,
Division of Biological Products, IVRI) and the non-tuberculous mycobacterial
(NTM) strains, including M. avium, M. intracellulare, M. fortuitum, M. chelonae, M.
gordonae, M. smegmatis, M. phlei and M. xenopi, were obtained from the
Tuberculosis Research Centre, Chennai, India. For the extraction of PCR
amplifiable DNA, a loop-full of mycobacterial growth was suspended in a microfuge
tube containing 400 μl of 1× TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). The
suspension was than subjected to boiling for 10 min followed by a brief
centrifugation and the supernatant was directly used as a template for PCR.
2.2. Species characterization
Frozen stocks of M. bovis and M. tuberculosis strains, maintained at the
Mycobacteria Laboratory at IVRI, were freshly grown on Lowenstein–Jensen (LJ)
medium. All M. tuberculosis and M. bovis strains were identified, as described
previously, by classical methods (Verma and Srivastava, 2001), as well as by PCR
based on allele-specific amplification of pncA and oxyR genes (Shah et al., 2002).
2.3. Primers and PCR conditions
The amplification primers for single tube m-PCR, designed in this study, were
based on the previously described sequences (Zumarraga et al., 1999). The
primers targeted a 229-bp sequence in M. bovis, which in the case of M.
tuberculosis, is interrupted at position 197 by a unique 12.7-kb fragment ORF
MTCY 227.28c encoding a hypothetical protein ‘Rv1506c’ (accession no. Z79701).
Oligonucleotide sequences of the primers used in the study were: the common
forward primer, CSB1 (5′-TTCCGAATCCCTTGTGA-3′), and two reverse primers,
including M. bovis-specific, CSB2 (5′-GGAGAGCGCCGTTGTA-3′), and M.
tuberculosis-specific, CSB3 (5′-AGTCGCGTGGCTTCTCTTTTA-3′).
The m-PCR reactions were performed in a total volume of 50 μl consisting of the
following: 5 μl of the template DNA, 25 pmol of each primer (CSB1, CSB2 and
CSB3), 200 μM of each dNTPs, 1.5 U of Taq DNA polymerase (Bangalore Genei,
Bangalore, India), 10 mM Tris–HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2 and 0.01%
(w/v) gelatin. The cycling parameters were: initial denaturation at 94 °C for 5 min,
followed by 30 three-step cycles, including denaturation at 94 °C for 1 min,
annealing at 52.3 °C for 1.5 min, extension at 72 °C for 1 min and a final extension
at 72 °C for 5 min. The amplification products were analyzed by electrophoresis on
1.5% (w/v) agarose gel and visualized by ethidium bromide fluorescence. The
unique amplification product of either 168 bp (M. bovis-specific) or 337 bp (M.
tuberculosis-specific) must be visualized using DNA from the respective species.
2.4. Specificity and sensitivity of multiplex PCR assay
To determine the specificity of the m-PCR assay, genomic DNAs from various nontuberculous mycobacteria mentioned above, were subjected to amplification by the
cited primer combinations. Serial 10-fold dilutions of DNA from both M. bovis AN5
and M. tuberculosis H37Rv strains were subjected to amplification by the designed
primers to determine the sensitivity of the m-PCR assay.
3. Results
3.1. Design of primers and optimization of m-PCR conditions
A common forward primer, CSB1, was designed to hybridize the 229-bp sequence
found in both M. bovis and M. tuberculosis, and complemented bases 50–66. The
M. bovis-specific reverse primer, CSB2, complements bases 217–202 of the 229bp sequence and is also expected to hybridize to the 229-bp sequence from both
organisms, but should generate a unique 168-bp PCR product in the case of M.
bovis only and not in M. tuberculosis. This is expected since the 229-bp sequence,
present in M. tuberculosis, is interrupted at position 197 by a unique 12.7-kb
fragment. In principle, in a PCR reaction, the 12.7-kb size insertion in M.
tuberculosis is beyond the amplification limits of Taq DNA polymerase and, hence,
cannot be amplified using primer CSB2. In contrast, M. tuberculosis-specific
reverse primer CSB3, which complements bases 23,729–23,708 of the 12.7-kb
fragment, is designed to hybridize to the 12.7-kb fragment and is expected to
generate a unique 337-bp PCR product specific to M. tuberculosis.
The m-PCR assay was initially tested with genomic DNA from standard strain M.
bovis AN5 and M. tuberculosis H37Rv. After several rounds of amplification and
testing different annealing temperatures (data not shown), adequate conditions
were found (see Section 2.4) to distinguish two species in a single reaction.
3.2. Rapid differentiation of M. bovis and M. tuberculosis by m-PCR
The m-PCR assay was applied to DNA from 35 M. tuberculosis and 20 M. bovis
strains, isolated from clinical cases of tuberculosis in human and animals. In all
cases, reaction mixtures with the template DNAs from M. bovis strains, including
M. bovis BCG, invariably showed a unique amplicon of 168 bp, while the reactions
with the DNAs from M. tuberculosis strain showed a unique amplicon of 337 bp
(Fig. 1). The m-PCR assay could not differentiate M. bovis BCG from those of M.
bovis non-BCG clinical strains, but the amplification results were consistent with
each strain of M. bovis and M. tuberculosis, irrespective of the source.
(36K)
Fig. 1. Ethidium bromide-stained 1.5% (w/v) agarose gel showing PCR products
amplified from representative strains of mycobacteria by m-PCR assay. Lanes: (M)
DNA molecular mass marker (100 bp ladder); (1) M. bovis AN5; (2) M. bovis BCG;
(3:) M. bovis 2/86 clinical isolate; (4) M. tuberculosis H37Rv; (5) M. tuberculosis
199/94 clinical isolate; (6) M. paratuberculosis; (7) M. avium; (8) M. smegmatis; (9)
M. chelonae; (10) M. fortuitum; (11) M. phlei; (12) M. intracellulare; (13) M. xenopi;
(14) M. gordonae.
3.3. Sensitivity and specificity of m-PCR
As determined by serial 10-fold dilutions of the template DNAs from both M. bovis
AN5 and M. tuberculosis H37Rv, the amplicons of 168 bp (M. bovis-specific) and
337 bp (M. tuberculosis-specific) could be visualized when amplifications were
performed with as little as 20 pg of chromosomal DNA (data not shown).
Templates from NTM strains produced no detectable PCR products justifying the
specificity of the technique (Fig. 1).
4. Discussion
The development of any species-specific PCR assay requires that the target gene
or DNA fragment be present in all the isolates from the particular species of
interest and be absent from all other unrelated species. In the past, the mtp-40
gene (M. tuberculosis-specific) and a 500-bp DNA fragment (M. bovis-specific)
were targeted to develop a PCR assay to differentiate between these two
pathogens (Del-portillo et al., 1991 and Rodriguez et al., 1995). However, the
specificity of these PCR methods was invalidated due to the findings that mtp-40
gene is also present in some M. bovis strains and the so-called M. bovis-specific
500-bp fragment is also present in some M. tuberculosis strains (Weil et al., 1996
and Shah et al., 2002).
In the present study, we designed a set of three PCR primers targeting the 229-bp
sequence polymorphism generated due to the presence of a 12.7-kb fragment in
the M. tuberculosis genome. This provided an improved method for definitive
detection of these two closely related species. The newly designed primers
correctly identified M. tuberculosis (337 bp) and M. bovis (168 bp) at species level
in a single tube reaction. The results obtained in this study indicated that the primer
sequences designed for the differentiation of closely related M. bovis and M.
tuberculosis are 100% specific. This m-PCR based on a 12.7-kb fragment
polymorphism does not differentiate M. bovis BCG isolate from those of M. bovis
non-BCG clinical strains. Our results, along with the findings of Zumarraga et al.
(1999), indicate that the presence of the 12.7-kb fragment is unique to M.
tuberculosis strains, as is its absence in M. bovis strains, tested from different
geographical areas and host species. The cross-reactivity of the designed primers
with other non-tuberculous mycobacteria was also excluded, as none of the NTM
strains tested showed presence of PCR amplicons. The m-PCR assay was
sensitive, as the amplicons of 168 bp and 337 bp could be visualized when the
PCR was performed with as little as 20 pg of genomic DNA from M. bovis and M.
tuberculosis strains, respectively.
Recently, two allele-specific PCR methods, based on allelic polymorphism in pncA
and oxyR genes, have also been described (De los Monteros et al., 1998).
Although these methods can differentiate M. bovis from M. tuberculosis cultures,
both assays require two differential amplifications to be performed in separate PCR
tubes for identification of a single isolate up to species level. A PCR assay was
described by Zumarraga et al. (1999), but it also required different sets of primers
and separate reactions for definitive identification of these two pathogens. Our mPCR assay, however, had a similar sensitivity (20 pg) as that of the allele-specific
PCR methods (De los Monteros et al., 1998), but with an advantage of being
simpler (requiring only a single tube reaction) and less expensive (saving reagents
required for an additional PCR reaction).
Although our data show 100% specificity, it is possible that analysis of larger
number of strains will identify organisms for which this polymorphism of 12.7 kb
fragment may break down. However, no reasonable number of strains can permit
us to rule out this possibility. Our analysis of polymorphism of the 12.7-kb fragment
in 55 strains studied here, along with the 20 strains studied by Zumarraga et al.
(1999), suggests that the organisms for which this association breaks down will be
relatively rare or confined to restricted geographical localities or host species.
Moreover, from an evolutionary point of view, it has been hypothesized that an
ancestor strain having the 12.7-kb fragment diverged toward M. tuberculosis and
M. bovis, and then M. bovis, lost this fragment (Zumarraga et al., 1999). Therefore,
it can be expected that the 12.7-kb fragment polymorphism is likely to be the most
stable genetic difference between these two pathogens. The results obtained using
the m-PCR assay designed in this study are, therefore, conclusive and indicate
that the use of m-PCR based on polymorphism in the 12.7-kb fragment reliably
differentiates M. bovis from M. tuberculosis. It appears that an m-PCR based on
the 12.7-kb region offers an excellent test for rapid and easy definitive identification
M. bovis or M. tuberculosis.
In conclusion, the m-PCR protocol described here is highly species-specific and
decreased the time needed for speciation of M. bovis and M. tuberculosis. This mPCR assay can be easily used as a routine monitoring tool in veterinary and
medical microbiology laboratories, especially in areas of endemicity, where bovine
and human TB coexist, and the separation of M. bovis from M. tuberculosis is
required for monitoring the spread of M. bovis to humans.
Acknowledgement
We are grateful to the Director, Indian Veterinary Research Institute, Izatnagar
243122, India, for providing necessary facilities to carry out this work.
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Fig. 1. Ethidium bromide-stained 1.5% (w/v) agarose gel showing PCR products
amplified from representative strains of mycobacteria by m-PCR assay. Lanes: (M)
DNA molecular mass marker (100 bp ladder); (1) M. bovis AN5; (2) M. bovis BCG;
(3:) M. bovis 2/86 clinical isolate; (4) M. tuberculosis H37Rv; (5) M. tuberculosis
199/94 clinical isolate; (6) M. paratuberculosis; (7) M. avium; (8) M. smegmatis; (9)
M. chelonae; (10) M. fortuitum; (11) M. phlei; (12) M. intracellulare; (13) M. xenopi;
(14) M. gordonae.