Differentiation of Mycobacterial Species by hsp65 Duplex PCR Followed
http://www.100md.com
《微生物临床杂志》
Department of Microbiology and Immunology, Cancer Research Institute and Liver Research Institute, College of Medicine, Seoul National University, Seoul 110-799
Mogam Biotechnology Research Institute Diagnostic Lab, Yongin 449-903
The Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul 137-140, South Korea
ABSTRACT
Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and differentiated all strains into their respective groups with 100% sensitivity and specificity. Furthermore, a duplex PCR-restriction analysis (duplex PRA) and a direct sequencing protocol were developed to differentiate NTM strains at the species and subspecies levels based on previously reported hsp65 DNA sequences (H. Kim et al., Int. J. Syst. Evol. Microbiol. 55:1649-1656, 2005) and then applied to 105 NTM clinical isolates. All NTM isolates were clearly differentiated at the species and subspecies levels by subsequent procedures (PRA or direct sequencing) targeting 515-bp NTM duplex PCR amplicons. Our results suggest that novel duplex PCR-based methods are sensitive and specific for identifying mycobacterial culture isolates at the species level.
INTRODUCTION
Of the validated species in the genus Mycobacterium, Mycobacterium tuberculosis is both the most common and the important pathogen, as it causes 2 million deaths and over 8 million cases of tuberculosis worldwide annually (1, 2, 3, 5). In addition to the multidrug-resistant strains of M. tuberculosis, NTM (nontuberculosis mycobacteria) infections also cause clinical problems. Because of the different pathogenic potentials and susceptibilities of NTM from M. tuberculosis, drugs of choice for the treatment of NTM infections differ (16, 20, 23). Thus, it is very important to differentiate M. tuberculosis strains and NTM strains during the early stage with a diagnostic procedure.
Instead of a culture-based identification scheme, which takes 4 to 6 weeks or longer for the identification of slowly growing mycobacteria, PCR and PCR-linked methods have been widely used to diagnose mycobacteria (6, 7, 14, 18). The insertion element, IS6110, which is uniquely found in multiple copies in M. tuberculosis complex (19), is most widely used for its detection and identification. Because of the increasing incidence of NTM infection, it is possible that methods which detect only M. tuberculosis fail to detect NTM. Thus, any method that can detect and identify M. tuberculosis and NTM strains simultaneously would be useful. For this purpose, multiplex PCR employing two or three different gene targets is frequently used. They could specifically detect and identify different species in the genus Mycobacterium (4, 13, 15, 22) or distinguish members of the M. tuberculosis complex (4, 8) in the routine diagnostic laboratory. Usually the 16S rRNA gene (22), groEL (8), and alpha-antigen gene (4) have been used as genus Mycobacterium-specific genes. The IS6110 insertion sequence (8), mpb70 gene (21), and mtp40 gene (4) have been used as M. tuberculosis complex-specific genes. However, some of these are not specific for M. tuberculosis. IS6110 was reported to cause false-negative (24) and false-positive results (10), and the mpt40 gene is not present in all M. tuberculosis strains (21). Duplex PCR targeting of a single gene, the RNA polymerase gene (11), has been developed for the differential identification of M. tuberculosis complex and NTM groups. However, this method has been reported to have problems associated with the short sequence length of the target gene. Therefore, a novel PCR method for the differential identification of M. tuberculosis complex and NTM groups and for the further species differentiation of NTM isolates is required.
Previously, we reported that sequence analysis of 604-bp hsp65 DNA is useful for differentiating mycobacterial species, and we introduced several signature nucleotides specific for M. tuberculosis and NTM strains (12). In the present study, we developed a novel duplex PCR method using these signature nucleotides. The devised method can differentiate M. tuberculosis and NTM strains by amplifying hsp65 DNAs of different sizes, i.e., of 195 bp and 515 bp, respectively, in a single PCR. Moreover, duplex PCR-restriction analysis and direct sequencing protocols for the further differentiation of NTM strains were also developed based on hsp65 DNA sequences. To demonstrate the usefulness of these protocols for mycobacterial identification, we applied them to 54 reference strains and 170 clinical mycobacteria isolates.
MATERIALS AND METHODS
Mycobacterial strains. Fifty-four reference strains (51 mycobacteria and 3 nonmycobacteria) (Table 1) and 170 clinical isolates were used in this study. The 54 reference strains and the 170 clinical isolates were provided by the Korean Institute of Tuberculosis (KIT). Clinical isolates were identified by growth characteristics and conventional biochemical tests (9) (Table 2). The results obtained by conventional biochemical tests were compared with those of duplex PCR-restriction analysis (PRA) and duplex PCR direct sequencing analysis, respectively. To identify NTM isolates, the sequences of hypervariable fragment A of 16S rRNA genes were also determined as previously reported (17). Briefly, 16S rRNA gene fragments were amplified using the forward primer 285 (5'-GAGAGTTTGATCCTGGCTCAG-3') and the reverse primer 264 (5'-TGCACACAGGCCACAAGGGA-3'), corresponding to bp 9 to 30 and 1046 to 1027 of Escherichia coli, respectively. Amplified products were directly sequenced with the sequencing primer 244 (5'-CCCACTGCTGCCTCCCGTAG-3').
Four heat-killed reference strains of Mycobacterium kansasii (subspecies II to V) and one strain of subspecies VI were kindly provided by Veronique Vincent (TB and Mycobacteria Lab, Institute of Pasteur, Paris) and Elvira Richter (Forschungszentrum Borstel, National Reference Center for Mycobacteria, Borstel, Germany).
DNA extraction. Chromosomal DNA was extracted by the bead beater-phenol extraction method (12). To disrupt Mycobacterium cell walls, a bacterial mixture containing phenol and glass beads was oscillated on a mini-bead beater. The aqueous phase was then transferred to a clean tube, and the DNA pellet was precipitated by adding isopropyl alcohol and then solubilized with 60 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Two microliters of purified DNA was used as a PCR template.
Duplex PCR. The devised duplex PCR used two hsp65 DNA fragments, 195 bp and 515 bp. These were specific for M. tuberculosis and NTM strains, respectively. Primers enabling the production of specific amplicons for each group were carefully designed using the signature nucleotides previously reported (12), especially the three consecutive signature nucleotides at codon 240 (Fig. 1). The primers were designed for specific nucleotides of M. tuberculosis members or NTM strains (each group) to be located at the 3' hydroxyl end. In a 20-μl PCR mixture tube (AccuPower PCR PreMix; Bioneer, Dae Jeon, Korea) containing 2 U of Taq polymerase, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and primers (10 pmol of 2TBF-2TBR and 20 pmol of MOTT3F-MOTT3R) were added, and the volume was adjusted to 20 μl. PCR was performed using initial denaturation at 95°C for 5 min, 30 amplification cycles (30 s at 95°C, 60 s at 72°C), and a final elongation at 72°C for 5 min (model 9600 thermocycler; Perkin-Elmer Cetus). Denaturation was extended to 15 min for the clinical isolates. The PCR products obtained were analyzed by agarose gel electrophoresis (2.5%). DNA bands were visualized by ethidium bromide staining and photographed.
Duplex PRA. A PRA algorithm for differentiating mycobacteria was constructed using MapDraw (version 3.14; DNASTAR, Madison, Wis.). After completing duplex PCR, PRA using 2 restriction enzymes, AvaII (TaKaRa) and HaeIII (TaKaRa), was performed to identify the 105 NTM strains that produced 515-bp hsp65 DNA. PRA reactions were performed as follows. Ten microliters of PCR products was transferred to a fresh microcentrifuge tube and digested with restriction enzymes according to the manufacturer's instructions. Following digestion, mixtures were electrophoresed on 3% agarose gel gels.
Direct sequencing. Purified PCR amplicons (515-bp) from 105 NTM clinical isolates were directly sequenced. During direct sequencing analysis, we used 515-bp PCR products as templates and MOTT3R, a reverse PCR primer, as a direct sequencing primer. Although approximately 470-bp sequences were obtained by direct sequencing, for reasons of simplicity, only 422-bp sequences (nucleotides 233 to 654 in the hsp65 gene of M. tuberculosis) were used as a target for direct sequencing analysis, as reported previously (12), for species differentiation (Fig. 1). An Applied Biosystems model 373A automatic sequencer and a BigDye terminator cycle sequencing kit (Perkin-Elmer Applied Biosystems) were used for the sequencing. For sequencing reactions, 60 ng of PCR-amplified DNA, 5 pmol of reverse primer (MOTT3R), and 4 μl of BigDye terminator v2.0 100 RR mix (Perkin-Elmer Applied Biosystems) were mixed. Contents were adjusted to a final volume of 10 μl by adding distilled water, and the reaction was run for 30 cycles of 10 s at 96°C, 5 s at 60°C, and 4 min at 60°C. Determined sequences (422 bp) were aligned with the sequences of 56 mycobacterial reference strains (GenBank accession no. AY299133 to AF299182 and AY373453 to AY373458) previously reported (12) by using the multiple-alignment algorithm in the MegAlign package (Windows version 3.12e; DNASTAR, Madison, Wis.) and were used for the species differentiation of NTM strains. A phylogenetic tree was constructed using the MEGA version 2.1 (13a), and a bootstrap analysis (1,000 repeats) was performed to evaluate the topology of the phylogenetic tree using Tsukamurella paurometabola KCTC 9821T as an out-group (AY373483).
RESULTS
Specificity and sensitivity of primer sets for DPCR. The specificity of each primer set (2TBF-2TBR and MOTT3F-MOTT3R) for duplex PCR was assessed by performing separate PCRs designed to specifically amplify the hsp65 DNAs (195 bp or 515 bp) of the 54 reference strains. When PCR using the M. tuberculosis-specific primer set (2TBF-2TBR) was applied using the same condition as duplex PCR (DPCR), 195-bp amplicons were observed from only 5 strains (Mycobacterium africanum, Mycobacterium bovis, M. bovis BCG, Mycobacterium microti, and M. tuberculosis) belonging to M. tuberculosis complex. When PCR using the NTM-specific primer set (MOTT3F-MOTT3R) was applied to the reference strains at the same annealing temperature, 515-bp DNAs were amplified from the other 49 strains (46 NTM strains plus 3 actinomycetes), except the M. tuberculosis complex (data not shown).
To evaluate affinities for target DNA and the sensitivities of each primer set, PCRs using each primer set were applied to the serially diluted DNAs of Mycobacterium avium (ATCC 25291) and M. tuberculosis (ATCC 27294). In PCRs using 2TBF-2TBR, the 195-bp amplicons were observed from 1 pg of M. tuberculosis DNA but not from even 100 ng of M. avium DNA. In PCRs using MOTT3F-MOTT3R, 515-bp amplicons were observed from 100 pg of M. avium DNA but not from 100 ng of M. tuberculosis DNA (data not shown).
Sensitivity of DPCR. The sensitivity of the developed DPCR method was evaluated in terms of the differential detection of M. avium (ATCC 25291) and M. tuberculosis (ATCC 27294) DNA in the serially diluted DNA samples. Amplification products were observed from 100 fg of M. tuberculosis DNA and 10 pg of M. avium DNA (Fig. 2).
Application of duplex PCR to reference and clinical isolates. When DPCR was applied to the 54 reference strains, 195-bp amplicons were produced from 5 strains of M. tuberculosis complex, whereas 515-bp amplicons were produced from the other 49 strains (Table 1; Fig. 3). To evaluate the usefulness of the DPCR method in a clinical setting, it was applied to 170 clinical isolates of mycobacteria, which had been cultured at KIT. Of these 170 culture isolates, 65 strains were identified as M. tuberculosis by the presence of 195-bp amplicons and the other 105 strains were identified as NTM by the presence of 515-bp amplicons. These results were completely concordant with those obtained by conventional culture testing (Table 3).
Identification of NTM strains by duplex PRA and direct sequencing. In the present study, for the species differentiation of strains identified as NTM by duplex PCR, two protocols using 515-bp DPCR amplicons were developed. First, a PRA algorithm capable of differentiating NTM strains by further digestion (AvaII and HaeIII) of duplex PCR amplicons was developed based on the hsp65 previously reported sequences of mycobacterial reference strains (Fig. 4) (12). To confirm the authenticity of this algorithm, we applied the developed duplex PRA to 54 reference strains and found that they produced the expected PRA pattern (Fig. 5A; Fig. 6). To evaluate the usefulness of duplex PRA for species differentiation in a clinical setting, this method was applied to the 105 clinical isolates identified as NTM by duplex PCR. Generally, the results obtained by this method were concordant with those obtained by both normal culture-based biochemical testing and partial 16S rRNA gene sequencing analysis (Table 4). The AvaII PRA of M. avium complex (MAC) strains, M. avium and Mycobacterium intracellulare, showed PRA patterns (X, 126, 72-bp) that differed from those of the other NTM strains (Fig. 4; Fig. 5A). Species differentiation between MACs was also possible using AvaII PRA alone (Fig. 5B).
Second, a direct sequencing protocol was developed that can differentiate NTM strains by analyzing 422-bp hsp65 sequences of DPCR amplicons. By inferring determined NTM sequences to the phylogenetic tree based on the previously reported hsp65 sequences of reference strains, all 105 NTM strains were successfully differentiated at the species or subspecies level (Table 4). Furthermore, the duplex PCR-based direct sequencing showed better resolution power than both normal culture-based biochemical testing and partial 16S rRNA gene sequencing analysis in separation between some rapidly growing species or differentiation of M. kansasii to the subspecies level (Fig. 7).
DISCUSSION
Multiplex PCR methods targeting several genes (4, 13, 15, 22) and duplex PCR targeting a single gene, the RNA polymerase gene (11), have been developed for the differential identification of M. tuberculosis complex and NTM strains. However, multiplex PCR methods have intrinsic problems due to the use of multiple target genes and lack of conservation in target genes. Although duplex PCR targeting the RNA polymerase gene could overcome problems caused by several target genes, it also has the problem in developing PRA or a direct sequencing protocol for the further differentiation of NTM strains due to the short length of the NTM amplicon (11). Species differentiation is not possible, though grouping is possible, among mycobacterial strains by the rpoB PRA method targeting 136-bp NTM amplicons. Furthermore, since the rpoB direct sequencing protocol targets only 76-bp sequences, except primer sequences, among the 136-bp NTM amplicons, differentiation between some species, such as Mycobacterium chelonae and Mycobacterium abscessus is not possible. Therefore, in the present study, to resolve some of the problems of previous methods, we developed a novel duplex PCR method targeting a single housekeeping gene, hsp65, and producing amplicons that were large enough for NTM differentiation.
Since in hsp65 duplex PCR, only a single gene, hsp65, is targeted irrespective of the M. tuberculosis complex or NTM species, the following were taken into account for successful amplification: (i) that the sizes of the amplicons of the two groups should be different enough to be discriminated in an agarose gel (195 versus 515 bp), (ii) that the optimal PCR annealing temperature of the respective primer sets should be nearly equal (60°C), and (iii) that the respective primer sets should be specific for target templates. In a previous study, we observed three consecutive signature nucleotides at codon 240 of HSP65 which are specific for M. tuberculosis complex strains (GGA) and NTM strains (CAG), suggesting that they could be effectively used for the development of M. tuberculosis or NTM detection methods. To construct TB-specific or NTM-specific primers, these consecutive signature nucleotides were used for one direction primer of each two-direction-primer pair (2TBF or MOTT3R) (Fig. 1). When PCR using the respective primer set was applied to the serially diluted DNA of M. tuberculosis or M. avium, it did not produce amplicons even at a nontarget DNA concentration of 1 μg (data not shown). As shown in a previous report (12), the present study also confirms that signature nucleotides of the hsp65 gene, specific for the M. tuberculosis complex and for the NTM strains, used for the development of the primer, are invariably constant. Thus, no false-positive or -negative results were obtained during the analysis of the 170 clinical isolates.
Since duplex PCR yields only one product irrespective of species for NTM, hsp65 DNAs of NTM (515-bp) can be further analyzed by PRA or by direct sequencing for species identification. Therefore, when duplex PRA and direct sequencing protocols were applied to the 105 NTM clinical isolates, all isolates were clearly differentiated at the species level. Furthermore, in M. kansasii, separation at the subspecies level and separation between M. abscessus and M. chelonae, which is not possible using rpoB duplex PCR-based protocols, were possible.
The other advantage of our hsp65 duplex PCR system is that a procedure required to separate MAC strains is straightforward, and these are the most frequently encountered and the most important NTM strains in the clinical setting. AvaII PRA alone can separate MACs from other NTM strains and for species differentiation between MACs. Therefore, in the general clinical setting, only DPCR-linked AvaII PRA, which can completely identify the three most clinically important strains, i.e., M. tuberculosis, M. avium, and M. intracellulare, is sufficient without further HaeIII PRA or a duplex PCR-based direct sequencing procedure.
In conclusion, duplex PCR targeting the hsp65 gene provides a rapid and reliable means of obtaining differential identification of M. tuberculosis complex and NTM in culture using only a single PCR. In addition to its advantages of simplicity and sensitivity, the method provides a clue for the differentiation of these two most important mycobacterial groups, which have different modes of infection and require different treatments. Furthermore, PRA and the sequencing of the duplex PCR products will add to the usefulness of the technique by allowing NTM identifications to be performed in clinical diagnostic laboratories.
ACKNOWLEDGMENTS
This study was supported by grant no. 09-2003-007-0 from the SNUH Research fund and in part by the BK21 Project for Medicine.
FOOTNOTES
Corresponding author. Mailing address: Department of Microbiology and Immunology, Cancer Research Institute and Liver Research Institute, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. Phone: 82 2 740 8316. Fax: 82 2 743 0881. E-mail: kbumjoon@snu.ac.kr.
Published ahead of print on 23 August 2006.
REFERENCES
Barnes, P. F., A. B. Bloch, P. T. Davison, and D. E. Sneider, Jr. 1991. Tuberculosis in patients with human immunodeficiency virus infection. N. Engl. J. Med. 324:1644-1649.
Bloom, B. R. 1992. Back to a frightening future. Nature (London) 358:538-539.
Bloom, B. R., and C. J. L. Murray. 1992. Tuberculosis: commentaty on a reemergent killer. Science 257:1055-1064.
Del Portillo, P., M. C. Thomas, E. Martinez, C. Maranon, B. Valladares, M. E. Patarroyo, and M. Carlos Lopez. 1996. Multiprimer PCR system for differential identification of mycobacteria in clinical samples. J. Clin. Microbiol. 34:324-328.
Dye, C., S. Scheele, P. Dolin, V. Pathania, and M. C. Raviglione. 1999. Global burden of tuberculosis. Estimated incidence, prevalence, and mortality by country. JAMA 282:677-686.
Eisenach, K. D., M. D. Sifford, M. D. Cave, J. H. Bates, and J. T. Crawford. 1991. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am. Rev. Respir. Dis. 144:1160-1163.
Folgueira, L., R. Delgado, E. Palenque, and A. R. Noriega. 1993. Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction. J. Clin. Microbiol. 31:1019-1021.
Herrera, E. A., O. Perez, and M. Segovia. 1996. Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by a multiplex-polymerase chain reaction. J. Appl. Bacteriol. 80:596-604.
Jenkins, P. A. 1992. The laboratory diagnosis of mycobacterial disease. Commun. Dis. Rep. CDR Rev. 2:101-103.
Kent, L., T. D. McHugh, O. Billington, J. W. Dale, and S. H. Gillespie. 1995. Demonstration of homology between IS6110 of Mycobacterium tuberculosis and DNAs of other Mycobacterium spp. J. Clin. Microbiol. 33:2290-2293.
Kim, B. J., S. K. Hong, K. H. Lee, Y. J. Yun, E. C. Kim, Y. G. Park, G. H. Bai, and Y. H. Kook. 2004. Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB). J. Clin. Microbiol. 42:1308-1312.
Kim, H., S. H. Kim, T. S. Shim, M. N. Kim, G. H. Bai, Y. G. Park, S. H. Lee, G. T. Chae, C. Y. Cha, Y. H. Kook, and B. J. Kim. 2005. Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int. J. Syst. Evol. Microbiol. 55:1649-1656.
Klemen, H., A. Bogiatzis, M. Ghalibafian, and H. H. Popper. 1998. Multiplex polymerase chain reaction for rapid detection of atypical mycobacteria and Mycobacterium tuberculosis complex. Diagn. Mol. Pathol. 7:310-316.
Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: Molecular evolutionary genetics analysis software. Arizona State University, Tempe, Ariz.
Manjunath, N., P. Shankar, L. Rajan, A. Bhargava, S. Saluja, and Shriniwas. 1991. Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis. Tubercle 72:21-27.
Mustafa, A. S., A. T. Abal, and T. D. Chugh. 1999. Detection of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria by multiplex polymerase chain reactions. East. Mediterr. Health J. 5:61-70.
Rosenzweig, D. Y. 1996. Nontuberculous mycobacterial disease in the immunocompetent adult. Semin. Respir. Infect. 11:252-261.
Springer, B., L. Stockman, K. Teschner, G. D. Roberts, and E. C. Bottger. 1996. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J. Clin. Microbiol. 34:296-303.
Sritharan, V., and R. H. Barker, Jr. 1991. A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR. Mol. Cell. Probes 5:385-395.
Thierry, D., A. Brisson-Noel, V. Vincent-Levy-Frebault, S. Nguyen, J. L. Guesdon, and B. Gicquel. 1990. Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis. J. Clin. Microbiol. 28:2668-2673.
Wagner, D., and L. S. Young. 2004. Nontuberculous mycobacterial infections: a clinical review. Infection 32:257-270.
Weil, A., B. B. Plikaytis, W. R. Butler, C. L. Woodley, and T. M. Shinnick. 1996. The mtp40 gene is not present in all strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 34:2309-2311.
Wilton, S., and D. Cousins. 1992. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl. 1:269-273.
Wolinsky, E. 1992. Mycobacterial diseases other than tuberculosis. Clin. Infect. Dis. 15:1-10.
Yuen, L. K., B. C. Ross, K. M. Jackson, and B. Dwyer. 1993. Characterization of Mycobacterium tuberculosis strains from Vietnamese patients by Southern blot hybridization. J. Clin. Microbiol. 31:1615-1618.(Hyun-Ju Kim, Ho-Suk Mun, Hong Kim, Eun-J)
Mogam Biotechnology Research Institute Diagnostic Lab, Yongin 449-903
The Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul 137-140, South Korea
ABSTRACT
Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and differentiated all strains into their respective groups with 100% sensitivity and specificity. Furthermore, a duplex PCR-restriction analysis (duplex PRA) and a direct sequencing protocol were developed to differentiate NTM strains at the species and subspecies levels based on previously reported hsp65 DNA sequences (H. Kim et al., Int. J. Syst. Evol. Microbiol. 55:1649-1656, 2005) and then applied to 105 NTM clinical isolates. All NTM isolates were clearly differentiated at the species and subspecies levels by subsequent procedures (PRA or direct sequencing) targeting 515-bp NTM duplex PCR amplicons. Our results suggest that novel duplex PCR-based methods are sensitive and specific for identifying mycobacterial culture isolates at the species level.
INTRODUCTION
Of the validated species in the genus Mycobacterium, Mycobacterium tuberculosis is both the most common and the important pathogen, as it causes 2 million deaths and over 8 million cases of tuberculosis worldwide annually (1, 2, 3, 5). In addition to the multidrug-resistant strains of M. tuberculosis, NTM (nontuberculosis mycobacteria) infections also cause clinical problems. Because of the different pathogenic potentials and susceptibilities of NTM from M. tuberculosis, drugs of choice for the treatment of NTM infections differ (16, 20, 23). Thus, it is very important to differentiate M. tuberculosis strains and NTM strains during the early stage with a diagnostic procedure.
Instead of a culture-based identification scheme, which takes 4 to 6 weeks or longer for the identification of slowly growing mycobacteria, PCR and PCR-linked methods have been widely used to diagnose mycobacteria (6, 7, 14, 18). The insertion element, IS6110, which is uniquely found in multiple copies in M. tuberculosis complex (19), is most widely used for its detection and identification. Because of the increasing incidence of NTM infection, it is possible that methods which detect only M. tuberculosis fail to detect NTM. Thus, any method that can detect and identify M. tuberculosis and NTM strains simultaneously would be useful. For this purpose, multiplex PCR employing two or three different gene targets is frequently used. They could specifically detect and identify different species in the genus Mycobacterium (4, 13, 15, 22) or distinguish members of the M. tuberculosis complex (4, 8) in the routine diagnostic laboratory. Usually the 16S rRNA gene (22), groEL (8), and alpha-antigen gene (4) have been used as genus Mycobacterium-specific genes. The IS6110 insertion sequence (8), mpb70 gene (21), and mtp40 gene (4) have been used as M. tuberculosis complex-specific genes. However, some of these are not specific for M. tuberculosis. IS6110 was reported to cause false-negative (24) and false-positive results (10), and the mpt40 gene is not present in all M. tuberculosis strains (21). Duplex PCR targeting of a single gene, the RNA polymerase gene (11), has been developed for the differential identification of M. tuberculosis complex and NTM groups. However, this method has been reported to have problems associated with the short sequence length of the target gene. Therefore, a novel PCR method for the differential identification of M. tuberculosis complex and NTM groups and for the further species differentiation of NTM isolates is required.
Previously, we reported that sequence analysis of 604-bp hsp65 DNA is useful for differentiating mycobacterial species, and we introduced several signature nucleotides specific for M. tuberculosis and NTM strains (12). In the present study, we developed a novel duplex PCR method using these signature nucleotides. The devised method can differentiate M. tuberculosis and NTM strains by amplifying hsp65 DNAs of different sizes, i.e., of 195 bp and 515 bp, respectively, in a single PCR. Moreover, duplex PCR-restriction analysis and direct sequencing protocols for the further differentiation of NTM strains were also developed based on hsp65 DNA sequences. To demonstrate the usefulness of these protocols for mycobacterial identification, we applied them to 54 reference strains and 170 clinical mycobacteria isolates.
MATERIALS AND METHODS
Mycobacterial strains. Fifty-four reference strains (51 mycobacteria and 3 nonmycobacteria) (Table 1) and 170 clinical isolates were used in this study. The 54 reference strains and the 170 clinical isolates were provided by the Korean Institute of Tuberculosis (KIT). Clinical isolates were identified by growth characteristics and conventional biochemical tests (9) (Table 2). The results obtained by conventional biochemical tests were compared with those of duplex PCR-restriction analysis (PRA) and duplex PCR direct sequencing analysis, respectively. To identify NTM isolates, the sequences of hypervariable fragment A of 16S rRNA genes were also determined as previously reported (17). Briefly, 16S rRNA gene fragments were amplified using the forward primer 285 (5'-GAGAGTTTGATCCTGGCTCAG-3') and the reverse primer 264 (5'-TGCACACAGGCCACAAGGGA-3'), corresponding to bp 9 to 30 and 1046 to 1027 of Escherichia coli, respectively. Amplified products were directly sequenced with the sequencing primer 244 (5'-CCCACTGCTGCCTCCCGTAG-3').
Four heat-killed reference strains of Mycobacterium kansasii (subspecies II to V) and one strain of subspecies VI were kindly provided by Veronique Vincent (TB and Mycobacteria Lab, Institute of Pasteur, Paris) and Elvira Richter (Forschungszentrum Borstel, National Reference Center for Mycobacteria, Borstel, Germany).
DNA extraction. Chromosomal DNA was extracted by the bead beater-phenol extraction method (12). To disrupt Mycobacterium cell walls, a bacterial mixture containing phenol and glass beads was oscillated on a mini-bead beater. The aqueous phase was then transferred to a clean tube, and the DNA pellet was precipitated by adding isopropyl alcohol and then solubilized with 60 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Two microliters of purified DNA was used as a PCR template.
Duplex PCR. The devised duplex PCR used two hsp65 DNA fragments, 195 bp and 515 bp. These were specific for M. tuberculosis and NTM strains, respectively. Primers enabling the production of specific amplicons for each group were carefully designed using the signature nucleotides previously reported (12), especially the three consecutive signature nucleotides at codon 240 (Fig. 1). The primers were designed for specific nucleotides of M. tuberculosis members or NTM strains (each group) to be located at the 3' hydroxyl end. In a 20-μl PCR mixture tube (AccuPower PCR PreMix; Bioneer, Dae Jeon, Korea) containing 2 U of Taq polymerase, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and primers (10 pmol of 2TBF-2TBR and 20 pmol of MOTT3F-MOTT3R) were added, and the volume was adjusted to 20 μl. PCR was performed using initial denaturation at 95°C for 5 min, 30 amplification cycles (30 s at 95°C, 60 s at 72°C), and a final elongation at 72°C for 5 min (model 9600 thermocycler; Perkin-Elmer Cetus). Denaturation was extended to 15 min for the clinical isolates. The PCR products obtained were analyzed by agarose gel electrophoresis (2.5%). DNA bands were visualized by ethidium bromide staining and photographed.
Duplex PRA. A PRA algorithm for differentiating mycobacteria was constructed using MapDraw (version 3.14; DNASTAR, Madison, Wis.). After completing duplex PCR, PRA using 2 restriction enzymes, AvaII (TaKaRa) and HaeIII (TaKaRa), was performed to identify the 105 NTM strains that produced 515-bp hsp65 DNA. PRA reactions were performed as follows. Ten microliters of PCR products was transferred to a fresh microcentrifuge tube and digested with restriction enzymes according to the manufacturer's instructions. Following digestion, mixtures were electrophoresed on 3% agarose gel gels.
Direct sequencing. Purified PCR amplicons (515-bp) from 105 NTM clinical isolates were directly sequenced. During direct sequencing analysis, we used 515-bp PCR products as templates and MOTT3R, a reverse PCR primer, as a direct sequencing primer. Although approximately 470-bp sequences were obtained by direct sequencing, for reasons of simplicity, only 422-bp sequences (nucleotides 233 to 654 in the hsp65 gene of M. tuberculosis) were used as a target for direct sequencing analysis, as reported previously (12), for species differentiation (Fig. 1). An Applied Biosystems model 373A automatic sequencer and a BigDye terminator cycle sequencing kit (Perkin-Elmer Applied Biosystems) were used for the sequencing. For sequencing reactions, 60 ng of PCR-amplified DNA, 5 pmol of reverse primer (MOTT3R), and 4 μl of BigDye terminator v2.0 100 RR mix (Perkin-Elmer Applied Biosystems) were mixed. Contents were adjusted to a final volume of 10 μl by adding distilled water, and the reaction was run for 30 cycles of 10 s at 96°C, 5 s at 60°C, and 4 min at 60°C. Determined sequences (422 bp) were aligned with the sequences of 56 mycobacterial reference strains (GenBank accession no. AY299133 to AF299182 and AY373453 to AY373458) previously reported (12) by using the multiple-alignment algorithm in the MegAlign package (Windows version 3.12e; DNASTAR, Madison, Wis.) and were used for the species differentiation of NTM strains. A phylogenetic tree was constructed using the MEGA version 2.1 (13a), and a bootstrap analysis (1,000 repeats) was performed to evaluate the topology of the phylogenetic tree using Tsukamurella paurometabola KCTC 9821T as an out-group (AY373483).
RESULTS
Specificity and sensitivity of primer sets for DPCR. The specificity of each primer set (2TBF-2TBR and MOTT3F-MOTT3R) for duplex PCR was assessed by performing separate PCRs designed to specifically amplify the hsp65 DNAs (195 bp or 515 bp) of the 54 reference strains. When PCR using the M. tuberculosis-specific primer set (2TBF-2TBR) was applied using the same condition as duplex PCR (DPCR), 195-bp amplicons were observed from only 5 strains (Mycobacterium africanum, Mycobacterium bovis, M. bovis BCG, Mycobacterium microti, and M. tuberculosis) belonging to M. tuberculosis complex. When PCR using the NTM-specific primer set (MOTT3F-MOTT3R) was applied to the reference strains at the same annealing temperature, 515-bp DNAs were amplified from the other 49 strains (46 NTM strains plus 3 actinomycetes), except the M. tuberculosis complex (data not shown).
To evaluate affinities for target DNA and the sensitivities of each primer set, PCRs using each primer set were applied to the serially diluted DNAs of Mycobacterium avium (ATCC 25291) and M. tuberculosis (ATCC 27294). In PCRs using 2TBF-2TBR, the 195-bp amplicons were observed from 1 pg of M. tuberculosis DNA but not from even 100 ng of M. avium DNA. In PCRs using MOTT3F-MOTT3R, 515-bp amplicons were observed from 100 pg of M. avium DNA but not from 100 ng of M. tuberculosis DNA (data not shown).
Sensitivity of DPCR. The sensitivity of the developed DPCR method was evaluated in terms of the differential detection of M. avium (ATCC 25291) and M. tuberculosis (ATCC 27294) DNA in the serially diluted DNA samples. Amplification products were observed from 100 fg of M. tuberculosis DNA and 10 pg of M. avium DNA (Fig. 2).
Application of duplex PCR to reference and clinical isolates. When DPCR was applied to the 54 reference strains, 195-bp amplicons were produced from 5 strains of M. tuberculosis complex, whereas 515-bp amplicons were produced from the other 49 strains (Table 1; Fig. 3). To evaluate the usefulness of the DPCR method in a clinical setting, it was applied to 170 clinical isolates of mycobacteria, which had been cultured at KIT. Of these 170 culture isolates, 65 strains were identified as M. tuberculosis by the presence of 195-bp amplicons and the other 105 strains were identified as NTM by the presence of 515-bp amplicons. These results were completely concordant with those obtained by conventional culture testing (Table 3).
Identification of NTM strains by duplex PRA and direct sequencing. In the present study, for the species differentiation of strains identified as NTM by duplex PCR, two protocols using 515-bp DPCR amplicons were developed. First, a PRA algorithm capable of differentiating NTM strains by further digestion (AvaII and HaeIII) of duplex PCR amplicons was developed based on the hsp65 previously reported sequences of mycobacterial reference strains (Fig. 4) (12). To confirm the authenticity of this algorithm, we applied the developed duplex PRA to 54 reference strains and found that they produced the expected PRA pattern (Fig. 5A; Fig. 6). To evaluate the usefulness of duplex PRA for species differentiation in a clinical setting, this method was applied to the 105 clinical isolates identified as NTM by duplex PCR. Generally, the results obtained by this method were concordant with those obtained by both normal culture-based biochemical testing and partial 16S rRNA gene sequencing analysis (Table 4). The AvaII PRA of M. avium complex (MAC) strains, M. avium and Mycobacterium intracellulare, showed PRA patterns (X, 126, 72-bp) that differed from those of the other NTM strains (Fig. 4; Fig. 5A). Species differentiation between MACs was also possible using AvaII PRA alone (Fig. 5B).
Second, a direct sequencing protocol was developed that can differentiate NTM strains by analyzing 422-bp hsp65 sequences of DPCR amplicons. By inferring determined NTM sequences to the phylogenetic tree based on the previously reported hsp65 sequences of reference strains, all 105 NTM strains were successfully differentiated at the species or subspecies level (Table 4). Furthermore, the duplex PCR-based direct sequencing showed better resolution power than both normal culture-based biochemical testing and partial 16S rRNA gene sequencing analysis in separation between some rapidly growing species or differentiation of M. kansasii to the subspecies level (Fig. 7).
DISCUSSION
Multiplex PCR methods targeting several genes (4, 13, 15, 22) and duplex PCR targeting a single gene, the RNA polymerase gene (11), have been developed for the differential identification of M. tuberculosis complex and NTM strains. However, multiplex PCR methods have intrinsic problems due to the use of multiple target genes and lack of conservation in target genes. Although duplex PCR targeting the RNA polymerase gene could overcome problems caused by several target genes, it also has the problem in developing PRA or a direct sequencing protocol for the further differentiation of NTM strains due to the short length of the NTM amplicon (11). Species differentiation is not possible, though grouping is possible, among mycobacterial strains by the rpoB PRA method targeting 136-bp NTM amplicons. Furthermore, since the rpoB direct sequencing protocol targets only 76-bp sequences, except primer sequences, among the 136-bp NTM amplicons, differentiation between some species, such as Mycobacterium chelonae and Mycobacterium abscessus is not possible. Therefore, in the present study, to resolve some of the problems of previous methods, we developed a novel duplex PCR method targeting a single housekeeping gene, hsp65, and producing amplicons that were large enough for NTM differentiation.
Since in hsp65 duplex PCR, only a single gene, hsp65, is targeted irrespective of the M. tuberculosis complex or NTM species, the following were taken into account for successful amplification: (i) that the sizes of the amplicons of the two groups should be different enough to be discriminated in an agarose gel (195 versus 515 bp), (ii) that the optimal PCR annealing temperature of the respective primer sets should be nearly equal (60°C), and (iii) that the respective primer sets should be specific for target templates. In a previous study, we observed three consecutive signature nucleotides at codon 240 of HSP65 which are specific for M. tuberculosis complex strains (GGA) and NTM strains (CAG), suggesting that they could be effectively used for the development of M. tuberculosis or NTM detection methods. To construct TB-specific or NTM-specific primers, these consecutive signature nucleotides were used for one direction primer of each two-direction-primer pair (2TBF or MOTT3R) (Fig. 1). When PCR using the respective primer set was applied to the serially diluted DNA of M. tuberculosis or M. avium, it did not produce amplicons even at a nontarget DNA concentration of 1 μg (data not shown). As shown in a previous report (12), the present study also confirms that signature nucleotides of the hsp65 gene, specific for the M. tuberculosis complex and for the NTM strains, used for the development of the primer, are invariably constant. Thus, no false-positive or -negative results were obtained during the analysis of the 170 clinical isolates.
Since duplex PCR yields only one product irrespective of species for NTM, hsp65 DNAs of NTM (515-bp) can be further analyzed by PRA or by direct sequencing for species identification. Therefore, when duplex PRA and direct sequencing protocols were applied to the 105 NTM clinical isolates, all isolates were clearly differentiated at the species level. Furthermore, in M. kansasii, separation at the subspecies level and separation between M. abscessus and M. chelonae, which is not possible using rpoB duplex PCR-based protocols, were possible.
The other advantage of our hsp65 duplex PCR system is that a procedure required to separate MAC strains is straightforward, and these are the most frequently encountered and the most important NTM strains in the clinical setting. AvaII PRA alone can separate MACs from other NTM strains and for species differentiation between MACs. Therefore, in the general clinical setting, only DPCR-linked AvaII PRA, which can completely identify the three most clinically important strains, i.e., M. tuberculosis, M. avium, and M. intracellulare, is sufficient without further HaeIII PRA or a duplex PCR-based direct sequencing procedure.
In conclusion, duplex PCR targeting the hsp65 gene provides a rapid and reliable means of obtaining differential identification of M. tuberculosis complex and NTM in culture using only a single PCR. In addition to its advantages of simplicity and sensitivity, the method provides a clue for the differentiation of these two most important mycobacterial groups, which have different modes of infection and require different treatments. Furthermore, PRA and the sequencing of the duplex PCR products will add to the usefulness of the technique by allowing NTM identifications to be performed in clinical diagnostic laboratories.
ACKNOWLEDGMENTS
This study was supported by grant no. 09-2003-007-0 from the SNUH Research fund and in part by the BK21 Project for Medicine.
FOOTNOTES
Corresponding author. Mailing address: Department of Microbiology and Immunology, Cancer Research Institute and Liver Research Institute, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. Phone: 82 2 740 8316. Fax: 82 2 743 0881. E-mail: kbumjoon@snu.ac.kr.
Published ahead of print on 23 August 2006.
REFERENCES
Barnes, P. F., A. B. Bloch, P. T. Davison, and D. E. Sneider, Jr. 1991. Tuberculosis in patients with human immunodeficiency virus infection. N. Engl. J. Med. 324:1644-1649.
Bloom, B. R. 1992. Back to a frightening future. Nature (London) 358:538-539.
Bloom, B. R., and C. J. L. Murray. 1992. Tuberculosis: commentaty on a reemergent killer. Science 257:1055-1064.
Del Portillo, P., M. C. Thomas, E. Martinez, C. Maranon, B. Valladares, M. E. Patarroyo, and M. Carlos Lopez. 1996. Multiprimer PCR system for differential identification of mycobacteria in clinical samples. J. Clin. Microbiol. 34:324-328.
Dye, C., S. Scheele, P. Dolin, V. Pathania, and M. C. Raviglione. 1999. Global burden of tuberculosis. Estimated incidence, prevalence, and mortality by country. JAMA 282:677-686.
Eisenach, K. D., M. D. Sifford, M. D. Cave, J. H. Bates, and J. T. Crawford. 1991. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am. Rev. Respir. Dis. 144:1160-1163.
Folgueira, L., R. Delgado, E. Palenque, and A. R. Noriega. 1993. Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction. J. Clin. Microbiol. 31:1019-1021.
Herrera, E. A., O. Perez, and M. Segovia. 1996. Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by a multiplex-polymerase chain reaction. J. Appl. Bacteriol. 80:596-604.
Jenkins, P. A. 1992. The laboratory diagnosis of mycobacterial disease. Commun. Dis. Rep. CDR Rev. 2:101-103.
Kent, L., T. D. McHugh, O. Billington, J. W. Dale, and S. H. Gillespie. 1995. Demonstration of homology between IS6110 of Mycobacterium tuberculosis and DNAs of other Mycobacterium spp. J. Clin. Microbiol. 33:2290-2293.
Kim, B. J., S. K. Hong, K. H. Lee, Y. J. Yun, E. C. Kim, Y. G. Park, G. H. Bai, and Y. H. Kook. 2004. Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB). J. Clin. Microbiol. 42:1308-1312.
Kim, H., S. H. Kim, T. S. Shim, M. N. Kim, G. H. Bai, Y. G. Park, S. H. Lee, G. T. Chae, C. Y. Cha, Y. H. Kook, and B. J. Kim. 2005. Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int. J. Syst. Evol. Microbiol. 55:1649-1656.
Klemen, H., A. Bogiatzis, M. Ghalibafian, and H. H. Popper. 1998. Multiplex polymerase chain reaction for rapid detection of atypical mycobacteria and Mycobacterium tuberculosis complex. Diagn. Mol. Pathol. 7:310-316.
Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: Molecular evolutionary genetics analysis software. Arizona State University, Tempe, Ariz.
Manjunath, N., P. Shankar, L. Rajan, A. Bhargava, S. Saluja, and Shriniwas. 1991. Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis. Tubercle 72:21-27.
Mustafa, A. S., A. T. Abal, and T. D. Chugh. 1999. Detection of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria by multiplex polymerase chain reactions. East. Mediterr. Health J. 5:61-70.
Rosenzweig, D. Y. 1996. Nontuberculous mycobacterial disease in the immunocompetent adult. Semin. Respir. Infect. 11:252-261.
Springer, B., L. Stockman, K. Teschner, G. D. Roberts, and E. C. Bottger. 1996. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J. Clin. Microbiol. 34:296-303.
Sritharan, V., and R. H. Barker, Jr. 1991. A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR. Mol. Cell. Probes 5:385-395.
Thierry, D., A. Brisson-Noel, V. Vincent-Levy-Frebault, S. Nguyen, J. L. Guesdon, and B. Gicquel. 1990. Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis. J. Clin. Microbiol. 28:2668-2673.
Wagner, D., and L. S. Young. 2004. Nontuberculous mycobacterial infections: a clinical review. Infection 32:257-270.
Weil, A., B. B. Plikaytis, W. R. Butler, C. L. Woodley, and T. M. Shinnick. 1996. The mtp40 gene is not present in all strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 34:2309-2311.
Wilton, S., and D. Cousins. 1992. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl. 1:269-273.
Wolinsky, E. 1992. Mycobacterial diseases other than tuberculosis. Clin. Infect. Dis. 15:1-10.
Yuen, L. K., B. C. Ross, K. M. Jackson, and B. Dwyer. 1993. Characterization of Mycobacterium tuberculosis strains from Vietnamese patients by Southern blot hybridization. J. Clin. Microbiol. 31:1615-1618.(Hyun-Ju Kim, Ho-Suk Mun, Hong Kim, Eun-J)