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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2004 Jun;42(6):2724–2732. doi: 10.1128/JCM.42.6.2724-2732.2004

Use of the hupB Gene Encoding a Histone-Like Protein of Mycobacterium tuberculosis as a Target for Detection and Differentiation of M. tuberculosis and M. bovis

S Prabhakar 1, A Mishra 1,†, A Singhal 1, V M Katoch 2, S S Thakral 3, J S Tyagi 1, H K Prasad 1,*
PMCID: PMC427828  PMID: 15184459

Abstract

The gene for histone-like protein (hupB [Rv2986c]) of Mycobacterium tuberculosis has been identified as a singular target which allows differentiation of two closely related mycobacterial species, namely, M. tuberculosis and M. bovis of the MTB complex, by a PCR assay. The N and S primer-generated PCR amplicons differed in M. tuberculosis and M. bovis; these amplicons were determined to be 645 and 618 bp, respectively. This difference was localized to the C-terminal part of the gene by using primers M and S. The C-terminal PCR amplicons of M. tuberculosis and M. bovis were determined to be 318 and 291 bp, respectively. The differences in the C-terminal portion of the gene were confirmed by restriction fragment length polymorphism analysis and sequencing. Sequence analysis indicated that in M. bovis there was a deletion of 27 bp (9 amino acids) in frame after codon 128 in the C-terminal part of the hupB gene. In the present study 104 mycobacterial strains and 11 nonmycobacterial species were analyzed for hupB gene sequences. Of the 104 mycobacterial strains included, 62 belonged to the MTB complex and 42 were non-MTB complex strains and species. Neither the hupB gene-specific primers (N and S) nor the C-terminal primers (M and S) amplify DNA from any other mycobacteria, making the assay suitable for distinguishing members of the MTB complex from other mycobacterial species, as well as for differentiating between members of the MTB complex, namely, M. tuberculosis and M. bovis.

Early and reliable detection of pathogenic mycobacteria in clinical samples is a major limitation in the control of human tuberculosis. At present, a battery of tedious tests (microbiological, biochemical, etc.) requiring more than several days or weeks are routinely used to identify clinical mycobacterial isolates. The criteria used for the differentiation of Mycobacterium tuberculosis and M. bovis have been colony morphology, nitrate reduction, niacin test, and sensitivity or resistance to pyrazinamide. Deviations from standard patterns in all of the above tests have been reported, making it virtually impossible to differentiate between M. bovis, M. tuberculosis, and M. africanum (12, 34, 38, 48). The high degree of variability in the phenotypic characteristics has made it important to develop reliable techniques to distinguish between members of the Mycobacterium tuberculosis and M. bovis (MTB) complex (22, 41). Techniques based on the amplification of mycobacterial DNA sequences by PCR have been introduced in many laboratories as a promising alternative rapid, sensitive, and specific detection of M. tuberculosis in clinical specimens (2, 7, 14).

Novel targets have been exploited for diagnostic purposes by using PCR, namely, the devR response regulator gene (42, 43), rRNA (5), selected chromosomal fragments (3, 18, 25, 29), genes coding for the 65-kDa heat shock protein (36), the 38-kDa protein antigen (44), the dnaJ gene (47), and insertion sequences such as IS6110, IS990, and IS1081 (1, 15, 16, 23); these are all examples of diverse targets that have been considered for PCR-based diagnostic approaches. Easy-to-use PCR kits targeting the rRNA gene for detection of M. tuberculosis are commercially available (Amplicor; Roche Molecular Systems, Branchburg, N.J.; GenProbe, Inc., San Diego, Calif.) (3, 51, 54), but it involves an additional step of DNA hybridization (3, 14). However, these gene targets are limited in their use for categorizing a Mycobacterium strain as belonging to the MTB complex or not. Alternate genetic markers and biochemical tests have been used to differentiate between M. tuberculosis, M. bovis, M. africanum, M. microti, and M. canetti (34). In addition to spoligotyping, the mtp40 gene sequence (28), pncA gene point mutation at position 169 (40), and polymorphism of the oxyR locus (46) have been reported as useful for identification of the members of the MTB complex. However, each of these targets has limitations. For example, the mtp40 gene and the insertion sequence IS6110 are not present in all strains of M. tuberculosis (15, 53).

It is important to identify species within the M. tuberculosis complex because of the zoonotic implications of bovine tuberculosis in developing countries and the potential for detecting patients with mixed pathogenic mycobacterial infections (27). M. tuberculosis, M. bovis, M. africanum, and M. canetti are recognized human pathogens. The clinical manifestation and treatment for each of these pathogens are identical, although the bovine bacillus is intrinsically resistant to pyrazinamide. Hence, diagnostic laboratories do not routinely identify these strains. Accurate identification of mycobacterial species is useful for detecting potentially hazardous public health infectious reservoirs and prevalence of mixed infection. In addition to identifying the source of infection, such differential diagnostic tests would benefit the formulation of relevant strategies for protection against pathogenic mycobacteria. Assays capable of differentiating and identifying pathogenic mycobacteria would be beneficial in the design of new and improved vaccines against human tuberculosis. Further, M. bovis is intrinsically resistant to pyrazinamide, which is used in the treatment of tuberculosis. Therefore, the availability of reliable molecular tools that distinguish closely related members of the MTB complex is of invaluable practical importance.

We report here a PCR assay for distinguishing M. tuberculosis and M. bovis by targeting the hupB (Rv2986c) gene in a single reaction. The strategy adopted is outlined in Fig. 1. Three primers—N, M, and S—were designed. Primer pair N-S amplified the entire hupB gene (Fig. 1B). The C-terminal part of the gene was selectively amplified by using primers M and S (Fig. 1C). The size differences in PCR products were observed to be reliable in distinguishing closely related mycobacterial species, namely, M. tuberculosis and M. bovis from other members of the MTB complex.

FIG. 1.

FIG. 1.

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Position of the hupB gene and primers used to generate PCR products. (A) Positions of the primers in the hupB sequence that were used to obtain the PCR products have been depicted. Primer pair N-S was specific for the hupB gene; internal primer pair M-S was specific for the C-terminal part of the hupB gene. (B and C) The ethidium bromide-stained amplification products of M. tuberculosis and M. bovis generated by using primers N and S (B) and primers M and S (C) were electrophoresed on nondenaturing 10% polyacrylamide gels. The 645- and 618-bp (B) and 318- and 291-bp (C) products are indicated. Lanes 1 and 4, 645 bp of hupB gene; lanes 6 and 9, 318 bp of the C-terminal part of the hupB gene amplification product obtained in M. tuberculosis H37Rv; lanes 2 and 5, 618 bp of the hupB gene; lanes 7 and 10, 291 bp of the C-terminal part of the hupB gene amplification product obtained in M. bovis AN5; lanes 3 and 8, 100-bp molecular weight markers.

MATERIALS AND METHODS

Bacterial strains.

The mycobacterial and nonmycobacterial strains used in the present study are listed in Table 1. In all, 104 mycobacterial strains were included in the study in addition to 11 nonmycobacterial species. Of the 104 mycobacterial strains, 62 were members of the MTB complex, (M. tuberculosis, 27 strains; M. bovis, 30 strains; M. microti, 3 strains; and 1 strain each of M. africanum and M. canetti). The details of the M. bovis strains included in the study are as follows: 14 strains were from infected cattle housed in the Central Military Veterinary Laboratory, Meerut, India, and 12 were cattle isolates obtained from the National Mycobacterial Repository, JALMA, Agra, India; 2 strains were from Argentina (J. D. A. van Embden, RIVM, Bilthoven, The Netherlands) and 2 were vaccine strains. All of the mycobacterial strains used in the present study have been identified at the National Mycobacterial Repository, JALMA, Agra, India. The tests include the cultivation of strains on Lowenstein-Jensen media with or without pyruvate. Species level identification of isolates was done by standard biochemical tests (niacin production, nitrate reduction, catalase and aryl sulfatase activity, Tween hydrolysis, thiopen-2-carboxylic acid hydrazide [TCH] sensitivity, etc.) as recommended by the Centers for Disease Control and Prevention (CDC), Atlanta, Ga., with appropriate controls. Further characterization of these isolates was done by PCR-restriction fragment length polymorphism (RFLP) analysis of the 16S-23S RNA spacer region. The nonmycobacterial strains included here were identified by using a panel of standard sugar fermentation tests at the Department of Microbiology, AIIMS, New Delhi, India.

TABLE 1.

Mycobacterial and nonmycobacterial species and strains used in the PCR assay

Species Strain no.b Source(s)a
Mycobacterium tuberculosis (human isolates) H37Rv, H37Ra, Erdman, P8473, P8497, C1207, C1084, 779634, ICC107, ICC120, ICC22, ICC238, ICC136, ICC37, ICC247, ICC16, ICC235, ICC145, ICC06, ICC11, ICC85, ICC95, CSU-17, CSU-27, CSU-20, CL125A, CL315 a, b, c, d, g, n
Mycobacterium bovis (cattle isolates) T11, AN5, IC378, IC379, IC380, IC381, IC382, ICC388, ICC391, 62, 66, 67, 117, 126, CL1, CL3, CL4, CL8, CL10, CL28, CL33, CL36, CL42, CL95, CL87, CL 113, CL320, CL370 d, o, p, q
Human vaccine strains Japan and Copenhagen o
Mycobacterium canetti 116 o
Mycobacterium africanum 81543 e, g
Mycobacterium microti OV254, T14, N5 d, f
Mycobacterium gastri TMC1456 b
Mycobacterium chelonae TMC191, J31, CL173, CL199, CL387, CL351 b
Mycobacterium vaccae IND123 b
Mycobacterium avium NCTC 8562, ICC192 d
Mycobacterium intracellulare TMC1302, N25, N8 d
Mycobacterium scrofulaceum TMC1302, MAC29 d
Mycobacterium gordonae TMC1324 d
Mycobacterium fortuitum 5J32, ICC420, ICC419, ICC417, ICC416, CL308 g, d, l
Mycobacterium smegmatis ATCC 27204, LR222, N18, CL310, CL381, CL462 b, d
Mycobacterium phlei ND124, N14, CL386 b
Mycobacterium kansasii 1201 c
Mycobacterium leprae Tissue biopsy d
Mycobacterium simiae IN7, CL357 d
Mycobacterium marinum CL370 d
Mycobacterium szulgai CL338 d
Mycobacterium terrae complex CL29, CL326 d
Mycobacterium triviale CL9 d
Mycobacterium flavescens CL209, CL350 d
Corynebacterium diphtheriae Clinical isolate h
Streptococcus haemolyticus Clinical isolate h
Staphylococcus aureus Clinical isolate h
Pseudomonas aeruginosa Clinical isolate h
Klebsiella pneumoniae Clinical isolate h
Nocardia asteroides MTCC274 i
Aspergillus fumigatus Soil isolate j
Aspergillus niger Soil isolate j
Candida albicans Clinical isolate k
Escherichia coli DH5α, BL21(DE3) m
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a

Sources: a, P. S. Murthy, UCMS, University of Delhi, New Delhi, India; b, N. K. Jain, NDTC, New Delhi, India; c, C. N. Paramasivan, TRC, Chennai, India; d, V. M. Katoch, JALMA, Agra, India; e, Y. M. Yates, Public Health Laboratory, Dulwich Hospital, London, United Kingdom; f, P. Draper, NIMR, Mill Hill, London, United Kingdom; g, Kathleen Eisenach, University of Arkansas, Little Rock; h, Department of Microbiology, AIIMS, New Delhi, India; i, Microbiological Type Culture Collection, IMTECH, Chandigarh, India; j, D. Shivkumar, Anna University, Chennai, India; k, Z. U. Khan, V.P. Chest Institute, New Delhi, India; l, Jack Crawfort, CDC; m, Gibco-BRL; n, Suman Laal, VA Medical Center, New York University School of Medicine, New York; o, J. D. A. van Embden, RIVM, Bilthoven, The Netherlands; p, Central Military Veterinary Laboratory, Meerut, India; q, Department of Pediatrics, AIIMS, New Delhi, India.

b

Numbers in boldface indicate human isolates.

Processing of bacilli for specificity analysis.

All of the mycobacterial and nonmycobacterial strains were grown on solid media (Lowenstein-Jensen slants, all mycobacterial species), Luria-Bertani agar (Escherichia coli), nutrient agar (Aspergillus niger, Nocardia asteroides, Pseudomonas aeruginosa, and Klebsiella pneumoniae) or blood agar (Corynebacterium diphtheriae and Streptococcus pneumoniae) were scraped with the help of sterile toothpicks and resuspended in sterile distilled water containing 0.1% Triton X-100. Resuspended bacilli were boiled at 100°C for 20 min, and an aliquot (2 μl) was used for PCR.

PCR analysis. (i) 23S ribosomal DNA (rDNA) target.

The primers C* (5′-GTGAGCGACGGGATTTGCCTAT-3′) and L* (5′-ACCACCCAAAACCGGATCGAT-3′) were used to detect the presence of DNA from organisms belonging to genus Mycobacterium. The expected size of the amplicon was 174 bp (11, 50).

(ii) hupB DNA target (international patent application no. PCT/IN03/00302).

The primers N (5′-GGAGGGTTGGGATGAACAAAGCAG-3′) and S (5′-GTATCCGTGTGTCTTGACCTATTTG-3′) were used to amplify hupB gene sequences. The expected size of the amplicon was 645 bp (Fig. 1).

(iii) C-terminal portion of the hupB gene.

The C-terminal portion of the hupB gene (applied for patent) was also amplified by using the internal primer M (5′-GCAGCCAAGAAGGTAGCGAA-3′) with primer S (5′-GTATCCGTGTGTCTTGACCTATTTG-3′). The expected amplicon was ∼318 bp (Fig. 1).

Each reaction (20 μl) contained 1.5 mM MgCl2, 0.5 μM concentrations of primers, 200 μM concentrations of deoxynucleoside triphosphates, 10 mM Tris-HCl (pH 8.8 at 25°C), 50 mM KCl, 0.08% Nonidet P-40, and 0.5 U of Taq DNA polymerase. The PCR for 23S rDNA and hupB DNA target was subjected to initial denaturation at 94°C for 10 min, and 35 cycles each of 1:30 min at 94°C, 1:30 min at 60°C, and 1:50 min at 72°C, followed by a final extension at 72°C for 30 min. The PCR for the C-terminal portion of the hupB gene was subjected to initial denaturation at 94°C for 10 min, and 35 cycles each of 1:0 min at 94°C and 1:30 min at 59°C, followed by a final extension at 72°C for 10 min. The products were analyzed on a 3.0% agarose gel-10% polyacrylamide gel and stained with ethidium bromide.

Southern hybridization.

The PCR amplicons resolved on the agarose gel were transferred onto nitrocellulose membrane (45). The blots were then hybridized with a α-32P-labeled hupB gene 645-bp probe generated by PCR with N and S primers and M. tuberculosis DNA.

RFLP.

DNA from different isolates of M. tuberculosis and M. bovis (listed in Tables 2 and 3) was amplified with (i) the N and S primers for the hupB gene and (ii) the M (internal primer, Fig. 1) and S primers for the C-terminal part of the hupB gene. hupB-amplified sequences were digested with HpaII and HaeIII restriction enzymes, and the products were analyzed on a 12% nondenaturing polyacrylamide gel. The gel was stained with ethidium bromide, and DNA fragments were visualized under UV light.

TABLE 2.

Representative results of hupB PCR assay with M. tuberculosis strains

Strain Source PCR-amplified product (645 bp/318 bp)
H37Rv ATCCa +/+
H37Ra ATCC +/+
Erdman ATCC +/+
779634 Human isolateb +/+
P8473 Human isolate +/+
P8497 Human isolate +/+
C1207 Human isolate +/+
C1084 Human isolate +/+
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a

Kathleen Eisenach, University of Arkansas, Little Rock.

b

C. N. Paramasivan, Tuberculosis Research Centre, Chennai, India.

TABLE 3.

Representative results of hupB PCR assay with M. bovis strains

Strain Source PCR-amplified product (618 bp/291 bp)
AN5 Cattle isolatea +/+
IC378 Cattle isolate +/+
IC379 Cattle isolate +/+
IC380 Cattle isolate +/+
IC381 Cattle isolate +/+
IC382 Cattle isolate +/+
117 Cattle isolate (Argentina)b +/+
126 Cattle isolate (Argentina) +/+
BCG Japanc +/+
BCG Copenhagen, Denmark +/+
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a

V. M. Katoch, JALMA, Agra, India.

b

J. D. A. van Embden, RIVM, Bilthoven, The Netherlands.

c

Paediatrics Department of AIIMS, New Delhi, India.

DNA sequencing analysis.

The PCR products were sequenced by the Sanger's dideoxy chain termination method (39) by using Sequenase version 2.0 sequencing kit, [35S]dATP, and forward and reverse universal M13 primers or internal primers of hupB according to the manufacturer's instructions. The DNA template was alkali denatured and annealed to the primers at −70°C for 1 h. The GC-rich mycobacterial DNA was mixed with 0.5 μg of single-strand binding protein prior to labeling. The protein was digested with proteinase K (0.1 μg) at 68°C for 20 min after termination of the labeling reaction. The reactions were electrophoresed on a 6% urea-polyacrylamide gel in 1× Tris-borate-EDTA at 70 W for a suitable time period. The gel was fixed with acetic acid (10%) and methanol (30%), dried, and autoradiographed. The PCR products obtained in standard strains and isolates were also sequenced commercially by Microsynth, Balgach, Switzerland.

RESULTS

23S rDNA-based PCR assay.

All of the mycobacterial strains included in the study tested with the 23S rDNA primers gave the predicted 174-bp amplicon. The 10 nonmycobacterial species included in the present study were negative for the assay (data not shown). These results are in agreement with those of an earlier report, indicating the specificity of these primers for the genus Mycobacterium (50).

Specificity of hupB-based PCR assay.

In order to check the specificity of the PCR assay using hupB gene as target, DNA from the mycobacterial and 10 nonmycobacterial species was used (Table 1). Representative data of the DNA extracted from standard strains and clinical isolates of M. tuberculosis and M. bovis (BCG) included for amplification with the hupB primers (N and S) are shown in Fig. 2. Only in the case of M. tuberculosis H37Rv, H37Ra, M. bovis BCG, and five clinical isolates of M. tuberculosis (Fig. 2A, lanes 1, 2, 3, 16, 17, 18, and 20, and B, lanes 1 and 12) was a PCR product obtained. No amplification was seen with DNA from M. microti, M. africanum, M. leprae, Mycobacterium avium-M. intracellulare-M. scrofulaceum (MAIS) complex, and other mycobacterial species (rapid and slow growers) or with DNA from C. diphtheriae and N. asteroides that, together with mycobacteria, make up the Corynebacterium-Mycobacterium-Nocardia (CMN) group. Amplification was also not seen in other nonmycobacterial species (Fig. 2B). The authenticity of the amplified product was confirmed by hybridization with α-32P-labeled hupB gene 645-bp probe generated by using N and S primers from M. tuberculosis (Fig. 2A′ and B′).

FIG. 2.

FIG. 2.

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Specificity analysis of hupB-based PCR assay. Amplification products were electrophoresed on 3.0% agarose gels. Their ethidium bromide staining (A and B) and hybridization profiles are shown in panels A′ and B′, respectively. The 645-bp product has been indicated. (A and A′) Lanes: 1, M. tuberculosis H37Rv; 2, M. tuberculosis H37Ra; 3, M. bovis BCG; 4, M. microti; 5, M. xenopi; 6, M. fortuitum; 7, M. phlei; 8, M. gordonae; 9, M. vaccae; 10, M. kansasii; 11, 100-bp molecular weight marker; 12, M. intracellulare; 13, M. avium; 14, M. scrofulaceum; 15, M. smegmatis; 16, M. tuberculosis P8497; 17, M. tuberculosis C1084; 18, M. tuberculosis 779634; 19, M. chelonei; 20, M. tuberculosis P8473; 21, M. gastri. (B and B′) Lanes: 1, M. tuberculosis 1207; 2, E. coli; 3, N. asteroides; 4, S. aureus; 5, P. aeruginosa; 6, S. faecalis; 7, S. aureus; 8, A. niger; 9, A. fumigatus; 10, C. albicans; 11, 100-bp molecular weight marker; 12, M. tuberculosis Erdman; 13, K. pneumoniae; 14, M. leprae; 15, M. africanum; 16, negative control. Hybridization in panels A′ and B′ was carried out with a 645-bp probe generated by using N and S primers.

C-terminal PCR assay.

PCR amplicons obtained from the DNA of M. bovis strains by using the hupB primers (N and S) (Fig. 3A, lanes 4 to 11) were marginally smaller compared to the PCR amplicons obtained from the M. tuberculosis strains (Fig. 3A, lanes 1 to 3). Using primers for the C-terminal part of the hupB gene the size difference in the PCR-amplified products of M. bovis and M. tuberculosis strains was clearly noticed (Fig. 3B). PCR amplicons obtained from the DNA of M. bovis strains by using the C-terminal hupB primers (M and S) (Fig. 3B, lanes 4 to 11) were smaller compared to PCR amplicons obtained from the M. tuberculosis strains (Fig. 3B, lanes 1 to 3). The results of the PCR assay with the two sets of primers are summarized in Tables 2 and 3. The 645- and 318-bp amplicons were obtained in all tested strains of M. tuberculosis. Comparative analysis on electrophoresis of the PCR products generated by the two sets of primer pairs namely, N-S and M-S, showed that the ability to distinguish between M. tuberculosis and M. bovis was best seen in the case of amplicons generated by the M and S primers electrophoresed in 10% nondenaturing polyacrylamide gels (Fig. 1B and C).

FIG. 3.

FIG. 3.

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Analysis of hupB gene PCR products and their RFLP. Ethidium bromide staining for hupB gene (A) and the C-terminal hupB gene (B) amplification products electrophoresed on agarose gels are shown. (A) Lanes: 1, M. tuberculosis H37Rv; 2, M. tuberculosis H37Ra; 3, M. tuberculosis Erdman; 4, M. bovis AN5; 5, M. bovis BCG (Japan); 6, M. bovis BCG (Copenhagen, Denmark); 7, M. bovis IC 378; 8, M. bovis IC379; 9, M. bovis IC380; 10, M. bovis IC381; 11, M. bovis IC382; 12, 100-bp molecular weight marker. (B) Lanes: 1 to 11, same as in panel A; 12, negative control; 13, 100-bp molecular weight marker. (C) RFLP polyacrylamide gel analysis of 645-bp PCR amplicon digested with HpaII (lanes 1 to 3) and HaeIII (lanes 5 to 8). Lanes: 1, M. tuberculosis H37Rv; 2, M. tuberculosis H37Ra; 3, M. bovis BCG; 4, 100-bp molecular weight marker; 5, M. tuberculosis H37Rv; 6, M. tuberculosis H37Ra; 7, M. bovis BCG; 8, M. bovis AN5. (D) Restriction digestion pattern of M. tuberculosis and M. bovis PCR products generated by using N and S primers with HpaII and HaeIII.

RFLP of PCR amplicons of the hupB gene derived from M. tuberculosis and M. bovis.

In order to confirm the observed marginal difference in the sizes of the PCR products generated with the N-S and M-S primer pairs, the amplified products were digested with HpaII and HaeIII (Fig. 3C and D). The digested products were analyzed on 10% nondenaturing polyacrylamide gels. Digestion of the N and S PCR products of M. tuberculosis and M. bovis with HpaII and HaeIII generates eight and seven fragments, respectively (Fig. 3D). Smaller bands were predicted for M. bovis (75- and 252-bp bands on digestion with HaeIII and HpaII, respectively) compared to the larger fragments obtained with M. tuberculosis (102- and 279-bp fragments). The HpaII fragments of 279 and 252 bp in M. tuberculosis and M. bovis are easily discernible. The difference in the smaller fragments of 102 and 75 bp generated by HaeIII digestion is not observable due to the presence of additional 29 bp in the N and S primer-generated PCR products of M. bovis and M. tuberculosis. All other fragments (six with HaeIII and seven with HpaII digests) of identical size in M. tuberculosis and M. bovis, ranging between 71 and 189 bp, were observed. The results, obtained with the amplicon generated in the C-terminal portion of the gene with M and S primers upon digestion with HpaII showed differences matching to those seen in case of the PCR product obtained with the N and S primers (results not shown). This indicates that the PCR-RFLP assay utilizing the PCR product obtained with either the hupB primers (N and S) or the C-terminal primers (M and S) and HpaII enzyme distinguished between M. tuberculosis and M. bovis.

Sequencing of PCR-amplified product.

The C-terminal PCR amplicons obtained with the M and S primers from DNA of standard strains of M. bovis and M. tuberculosis, including nine isolates of M. bovis (CL1, CL3, CL4, CL8, CL10, CL33, IC380, IC381, and An5) and one M. tuberculosis isolate (CL42) derived from cattle were sequenced. Sequence analysis indicated that in M. bovis there was a deletion of 27 bp (nine amino acids) in frame after the 128th codon in the C-terminal part of the gene (Fig. 4). The histone-like gene sequence of M. bovis has been submitted to the NCBI database (accession no. Y18421).

FIG. 4.

FIG. 4.

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Nucleotide sequence alignment of hupB gene of M. tuberculosis and M. bovis. The nucleotide sequence of the C-terminal region (369 to 418 bp) of the hupB gene of standard strains of M. tuberculosis and M. bovis and clinical mycobacterial strains has been aligned by using GCG software. A deletion of 27 bp was seen in hupB sequence of all M. bovis strains. The nine deleted amino acids (KAATKAPAR) between 385 and 411 bp with respect to M. tuberculosis are shown in single letter code on the first line. Numbers refer to nucleotide position in hupB (Rv2986c) and M. bovis (accession no. Y18421). Numbers in brackets refer to the PCR amplicon sizes. The strain numbers are given on the left. M. bovis: CL 33, CL1, CL10, CL3, CL4, CL8, IC380, IC381, and AN5; M. tuberculosis, CL42.

The 27-bp difference in the PCR-amplified products of the hupB gene of M. tuberculosis and the histone-like gene of M. bovis can be determined by using a single set of PCR primers. The most appropriate are the C-terminal primers (M and S) compared to the hupB gene primers (N and S) to distinguish between M. tuberculosis and M. bovis. The PCR products electrophoresed on polyacrylamide gels permit PCR product size discrimination, negating the requirement of RFLP to be carried out routinely (Fig. 1B and C).

DISCUSSION

In earlier studies carried out in our laboratory, the M. tuberculosis hupB gene (Rv2986c, accession no. P95109) and its protein were identified and characterized (37). The ability of the protein to bind DNA and induce lymphoproliferation and antibodies in tuberculosis patients has been reported earlier (37). In the present study we describe a PCR assay that precisely identifies closely related mycobacteria belonging to the MTB complex. The hupB gene target is useful in the differentiation of M. tuberculosis from M. bovis species from among other members of the tuberculosis complex, mycobacterial and nonmycobacterial species tested. The assay would prove useful especially in developing countries with a high incidence of infected livestock (21). M. bovis has been known to spread to humans from infected cattle (zoonotic tuberculosis) by aerosol or by consumption of contaminated dairy products (10, 33, 52). Bovine tuberculosis has been on the increase in developed countries (www.defra.gov.uk/animalh) and continues to occur in developing countries (10, 21). The epidemiological impact of bovine tuberculosis on human health has not been assessed and is a major lacuna in developing countries. However, with reports of tuberculosis due to M. bovis in AIDS patients (6, 35) and with increasing incidence of tuberculosis globally, rapid and reliable diagnostic assays are required not only for detection but also for identification of the pathogenic mycobacteria in clinical samples. This is essential for the prompt diagnosis, treatment, and control of tuberculosis. Despite the inherent natural resistance of M. bovis to pyrazinamide, this has generally not been a major difficulty in treatment administered to patients, since the bovine tubercle bacilli have been shown to be sensitive to other commonly used chemotherapeutic agents. However, identification of the currently difficult-to-characterize human pathogenic species of the tuberculosis complex in clinical samples will bring into focus (i) the incidence of these potential pathogenic mycobacteria in clinical samples, (ii) a comparative analysis of factor(s) that contribute to pathogenesis of these closely related species with the classical tubercle bacilli, and (iii) the critical components in a vaccine(s) required for prevention and control of human tuberculosis caused by pathogenic mycobacteria such as M. tuberculosis and M. bovis. It has been documented that the currently used vaccine, namely, BCG, for the prevention of human tuberculosis exhibits large variation in its efficiency to generate protective immunity in immunized individuals (17). This variation in immunizing efficiency has been attributed to the observed deletions resulting in genetic differences in BCG vaccine strains used for human immunization (4). However, this variation may well be due to vaccine strains being effective against particular human pathogenic mycobacteria and ineffective against others (48, 49). Identification of human mycobacterial pathogens in clinical specimens has been limited by the currently available techniques for precise and early identification of mycobacteria.

On the basis of the observations described here, we propose that the hupB gene can be used as a target for specific identification and to differentiate between M. tuberculosis and M. bovis by PCR. The precise differences in the PCR amplicons can be confirmed by RFLP assay. The difference in the PCR product size between M. bovis and M. tuberculosis isolates was ascribed to differences in the C-terminal portion of the hupB gene. This difference was stable and detected in standard, as well as in all locally isolated strains of M. bovis and M. tuberculosis included in the present study. Single-step PCR procedures to differentiate M. bovis from M. tuberculosis by using IS6110 (9, 24) alone or in association with mtp40 gene have yielded discrepant results (9, 13, 28). Further, it has been shown that mpt40 is not present in all M. tuberculosis strains and thus may not be useful for differentiating M. tuberculosis and M. bovis strains (53). The sen X3-regX3 intergenic region has been proposed as a target sequence for differentiating members of the MTB complex from other mycobacteria. However, there are limitations in the use of this target region since it cannot identify members of the MTB complex, although BCG could be discerned from related strains (30). The primers used in the present study for amplification of the hupB gene were specific for M. tuberculosis and M. bovis. This was confirmed by hybridization with α-32P-labeled PCR product obtained by using the primers N and S (Fig. 2A′ and B′) since no other amplification was obtained with any other template DNA that could have been missed by ethidium bromide staining alone.

Comparative DNA microarrays and genome hybridization arrays have detected 14 regions (RD1 to RD14) that are absent in M. bovis BCG Pasteur strain relative to M. tuberculosis H37Rv (4, 8, 20, 26) and two deletions specific for M. bovis BCG (4). The hupB gene is not located in the RD loci. The sequencing project has revealed that the genome of M. bovis is >99.9% identical to M. tuberculosis. Thus far, no single target sequence has provided 100% sensitivity and a total absence of false-positive results when used alone (29). However, hupB is present in every isolate of M. tuberculosis and M. bovis we have analyzed thus far (total tested = 57). We propose that the hupB-based PCR assay could be exploited as a target for the specific detection of M. tuberculosis and for the differentiation of M. tuberculosis and M. bovis strains. This assay has been utilized for the direct detection of M. tuberculosis and M. bovis in cattle and human samples (unpublished data). Upon analyzing the sequence of histone-like gene of M. bovis, it was seen that there was a deletion of 27 bp corresponding to nine amino acids: 618 bp compared to the amplicon obtained in M. tuberculosis. Correspondingly, the C-terminal PCR products obtained with primers M and S were 318 bp in M. tuberculosis and 291 bp in M. bovis, respectively. Thus, the hupB gene was found to be 645 bp (214 amino acids) in M. tuberculosis and 618 bp (205 amino acids) in M. bovis. Matsumoto et al. (31, 32) had also reported the hupB gene (MDP1) of M. bovis (BCG, Tokyo) to be 205 amino acids (accession no. AB013441). This finding contrasts with the hupB gene sequence (accession no. NP_856655) of M. bovis found by Garnier et al. (19).

The assay described here has the potential to be used as a diagnostic assay for the detection and identification of pathogenic mycobacteria in clinical samples and is a useful adjunct in quality control in the screening of dairy and meat products.

Acknowledgments

We thank C. N. Paramasivan, K. Eisenach, P. S. Murthy, N. K. Jain, M. Yates, P. Draper, P. Aggrawal, Z. U. Khan, J. Crawford, and J. D. A. van Embden for kindly providing the various M. tuberculosis and M. bovis isolates and strains.

This study was supported by DBT (Department of Biotechnology) India. S.P. and A.S. were a recipients of an SRF fellowship from the CSIR (Council of Scientific and Industrial Research) and UGC (University Grant Commission) of India.

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