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Journal of Clinical Microbiology, November 2000, p. 4080-4085, Vol. 38, No. 11
Institute for Biomedical Research,
SJ-Hightech Co., Ltd.,1 and Departments
of Biochemistry,2 Clinical
Pathology,3 and Internal
Medicine,4 College of Medicine, Pusan
National University, Pusan, and Institute of Clinical Research,
National Masan Tuberculosis Hospital, Masan,5
Korea
Received 29 March 2000/Returned for modification 8 May
2000/Accepted 10 July 2000
We evaluated the usefulness of PCR assays that target the internal
transcribed spacer (ITS) region for identifying mycobacteria at the
species level. The conservative and species-specific ITS sequences of
33 species of mycobacteria were analyzed in a multialignment analysis.
One pair of panmycobacterial primers and seven pairs of mycobacterial
species-specific primers were designed. All PCRs were performed under
the same conditions. The specificities of the primers were tested with
type strains of 20 mycobacterial species from the American Type Culture
Collection; 205 clinical isolates of mycobacteria, including 118 Mycobacterium tuberculosis isolates and 87 isolates of
nontuberculous mycobacteria from 10 species; and 76 clinical isolates
of 28 nonmycobacterial pathogenic bacterial species. PCR with the
panmycobacterial primers amplified fragments of approximately 270 to
400 bp in all mycobacteria. PCR with the M. tuberculosis
complex-specific primers amplified an approximately 120-bp fragment
only for the M. tuberculosis complex. Multiplex PCR with
the panmycobacterial primers and the M. tuberculosis
complex-specific primers amplified two fragments that were specific for
all mycobacteria and the M. tuberculosis complex,
respectively. PCR with M. avium complex-, M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, and
M. szulgai-specific primers amplified specific fragments
only for the respective target organisms. These novel primers can be
used to detect and identify mycobacteria simultaneously under the same
PCR conditions. Furthermore, this protocol facilitates early and
accurate diagnosis of mycobacteriosis.
It is estimated that there are 8 million cases of tuberculosis (TB), causing 2.5 million deaths per
year, worldwide, making TB the foremost cause of death due to
infection. Mycobacterial infections due to nontuberculous mycobacteria
(NTM), such as the Mycobacterium avium complex (MAC),
M. fortuitum, and M. chelonae, are also on the
increase (20). The increasing number of mycobacterial infections has made it clinically important to quickly identify mycobacteria at the species level. The diagnosis of pathogenic versus
nonpathogenic species not only has epidemiological implications but
also is relevant for patient management (1).
PCR has proven to be a very useful tool for the rapid diagnosis of
infectious diseases, including mycobacteriosis. Many of the PCR assays
used for detecting mycobacteria involve species-specific primers
targeting the 16S rRNA, hsp65, 32-kDa protein genes, or the
internal transcribed spacer (ITS) and detect only a single or a limited
number of mycobacterial species (10, 13). The ITS between
the l6S and 23S rRNA genes is approximately 270 to 360 bp but varies in
size from species to species. It is considered to be a suitable target
for probes with which additional phylogenetic information can be
derived (15). Furthermore, the ITS is suitable for
differentiating species of mycobacteria and potentially can be used to
distinguish clinically relevant subspecies (12). With
respect to mycobacteria, both the high level of spacer sequence variation and the good reproducibility of ITS sequencing suggest the
applicability of this approach. The purposes of this study were to
design genus-specific and species-specific primers and to determine the
PCR conditions for the simple and accurate detection of clinically
important mycobacterial species.
Primer design.
Conservative and polymorphic ITS sequences of
mycobacteria were sought in a multialignment of the ITS regions of 33 mycobacterial species using CLUSTAL-W (http://genome.kribb.re.kr)
(Table 1). The ITS sequences of 31 mycobacterial species were obtained from GenBank. The ITS regions of
M. fortuitum and M. chelonae were cloned and sequenced, as there were no sequence data in GenBank, even
though these species are frequently isolated from clinical specimens
(20). The sequence identity between the designed primers and
the mycobacterial ITS regions was analyzed with a BLAST search (http://www.ncbi.nlm.nih.gov). Based on the multialignment analysis data, a mycobacterial genus-specific primer pair and seven
species-specific primer pairs were designed (Table
2). A set of primers, ITS-F and mycom-2,
was used to amplify partial ITS regions in mycobacteria. The two
primers were designed from the highly conserved region on the basis of
16S rRNA and ITS sequences of mycobacteria, respectively (3). Species-specific primers were designed from the
polymorphic regions of ITS sequences of mycobacteria.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Detection and Identification of Mycobacteria by Amplification of
the Internal Transcribed Spacer Regions with Genus- and
Species-Specific PCR Primers
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains used for sequence analysis and alignment of
the ITS regions
TABLE 2.
Genus- and species-specific primers designed for the
detection of mycobacteria
Bacterial strains.
The type strains of 20 mycobacterial
species from the American Type Culture Collection and 1l8 M. tuberculosis and 87 NTM clinical isolates were used in this study.
M. tuberculosis clinical isolates were randomly selected
from the stored strains at the mycobacterial laboratories of Pusan
National University Hospital and National Masan Tuberculosis Hospital.
NTM (clinical isolates) were identified by conventional methods and
kindly provided by the Korean National Tuberculosis Association, which
is the reference laboratory for tuberculosis diagnosis in eastern Asia.
In the case of discrepancies between traditional and PCR methods, we confirmed our results by sequence analysis of the ITS. Fifty-five clinical isolates of nonmycobacterial pathogens were included to
confirm the specificity (Table 3). These
isolates were identified biochemically or with commercial kits, such as
API (Biomerieux, Marcy l'Etoile, France) and Vitek (BioMerieux Vitek,
Hazelwood, Mo.) kits.
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Preparation of genomic DNA and PCR. All the mycobacteria were subcultured on Ogawa media, and nonmycobacteria were subcultured on blood agar plates. DNA was prepared from freshly grown colonies using an InstaGene matrix kit (Bio-Rad). PCR was performed with each pair of genus-specific primers and species-specific primers under the same conditions. Multiplex PCR was performed using panmycobacterial and M. tuberculosis complex-specific primers in the same reaction tube. The primers were synthesized at a 50-nmol concentration (reagents were supplied by BioBasic Inc.). We tested the PCR conditions by adding various concentrations of tetramethylammonium chloride (TMAC), dimethyl sulfoxide, and glycerol to reduce nonspecific amplifications; finally, the following conditions were chosen. The constituents of the PCR mixtures were as follows: 500 mM KCl, 100 mM Tris HCl (pH 9.0), 1% Triton X-100, 0.2 mM each deoxynucleoside triphosphate (dATP, dGTP, dTTP, and dCTP), 1.5 mM MgCl2, 10 µM TMAC, 10 pmol of each primer, and 1 U of Taq DNA polymerase (BioBasic). Each reaction was carried out for 5 min at 94°C; 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C; and 10 min at 72°C. The products were electrophoresed in 1.5% agarose gels. For multiplex PCR for the detection of mycobacteria and M. tuberculosis, the primer concentration was optimized by mixing the ITS-F (10 pmol), mycom-2 (30 pmol), and TBF (20 pmol) primers together.
Nucleotide sequence accession numbers. The sequences determined in this study were deposited in GenBank under accession numbers AF144326 and AF144327.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Designed primers.
The ITS sequence has two conserved regions
and several polymorphic regions in the 33 mycobacteria examined,
including M. fortuitum and M. chelonae (Fig.
1). One pair of genus-specific and seven pairs of species-specific primers for mycobacteria were designed after
consideration of product size and melting temperature (Table 3). The pair of panmycobacterial primers
was expected to amplify specific fragments of 270 to 400 bp from
species to species. Each pair of M. tuberculosis
complex-, MAC-, M. fortuitum-, M. chelonae-, M. gordonae-,
M. scrofulaceum-, and M. szulgai-specific primers was
expected to amplify specific fragments of 121, 144, 223, 93, 152, 99, and 105 bp in the respective target mycobacteria.
|
Detection of mycobacteria.
For all the tested type strains of
mycobacteria, the specific fragments were amplified in the PCR
with the ITS-F and mycom-2 primers. The amplicons were
approximately 270 to 400 bp in size, as expected (Fig.
2). For 118 M. tuberculosis
and 87 NTM clinical isolates, the expected specific fragments were
amplified with genus-specific primers (ITS-F and mycom-2). Nonspecific
amplicons were not seen in any of the nonmycobacterial pathogens tested (Table 3).
|
Identification of mycobacteria.
PCR with the M. tuberculosis complex-specific primers, TBF and TBR, amplified an
approximately 121-bp fragment only in the M. tuberculosis
and M. bovis type strains (Fig.
3). As shown in Fig. 3, PCR with each
pair of NTM-specific primers amplified the specific fragment of the
expected size only in strains of the target organism. All NTM clinical
isolates were tested with each set of species-specific primers, and the
specificity was confirmed.
|
Multiplex PCR.
PCR with panmycobacterial and M. tuberculosis complex-specific primers in the same tube amplified
the two expected fragments, approximately 274 and 121 bp in size, in
M. tuberculosis and M. bovis type strains and one
panmycobacterial fragment in each of the NTM type strains (Fig.
4). The same result was also obtained for
clinical isolates of M. tuberculosis and NTM. No fragment was found by PCR for nonmycobacterial pathogens.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Rapid identification of species of mycobacteria is an important factor for a successful diagnosis of mycobacteriosis. It facilitates selection of the appropriate drug therapy. However, it is not easy to identify species of mycobacteria, especially NTM. The Accuprobe culture confirmation kit (Gen-Probe Inc., San Diego, Calif.), one of the commercially available methods for mycobacterial identification, can be used for only a limited number of mycobacterial species (13). Furthermore, the kit is relatively expensive. High-performance liquid chromatography can also be used for mycobacterial identification, and its operating cost is low. However, the equipment is expensive (7).
Currently, the widely accepted strategy formulated to improve methods for mycobacterial strain identification includes analysis of the gene encoding 16S rRNA (15). Other target genes have been proposed for the identification of mycobacteria by PCR-based sequencing. They included the spoligotyping (spacer oligonucleotide typing) region (2, 21), the 32-kDa protein gene (16), the dnaJ gene (18), the superoxide dismutase gene (22), the 65-kDa heat shock protein gene (hsp65) (14, 19), and the RNA polymerase gene (rpoB) (11). Each technique has several advantages and disadvantages. For example, an excessive degree of variability, such as that found in the hsp65 gene, may be undesirable, because such variability or instability of species-specific signatures will make the development of reliable probes that cover all strains within a species impossible (15). 16S rDNA sequences do not vary greatly within a species, and they are identical in some species (20). Molecular typing by 16S rRNA sequence determination is not only more rapid but also more accurate than traditional typing (17).
Comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. For M. tuberculosis, rifampin resistance can be simultaneously determined (11).
This study demonstrated that an ITS-based PCR method has a high degree of sensitivity and specificity for the detection and identification of medically important species of mycobacteria. Glennon et al. (8) first speculated on its utility for the diagnosis of TB. The ITS sequence between the 16S rRNA and 23S rRNA genes, which is more variable than the 16S rRNA gene itself, has been shown to be species specific in many microorganisms (9). However, there is little between-species variation in the length of the spacer, which ranges from 235 nucleotides for M. xenopi to 285 nucleotides for M. gastri, a slowly growing mycobacterium. The spacer sequences of slowly growing species are approximately 75 nucleotides shorter than those of rapid growers. Frothingham and Wilson (6) demonstrated intraspecies sequence polymorphisms in 4 of 11 species. M. gastri and M. avium each were split into two distinct sequevars (sequence variations), designated Mga-A and Mga-B and Mav-A and Mav-B, respectively, based on the nomenclature proposed by Frothingham and Wilson (6).
It was possible to develop genus- and species-specific primers because the ITS has two conserved regions and several polymorphic regions in the 33 mycobacterial species. The two conserved regions are close to each other within the mycobacterial ITS (Fig. 1). The primers mycom-1 and mycom-2 were designed from these two conserved sequences. The amplicons for the primer set mycom-1 and ITS-R were expected to be approximately 120 to 250 bp long. In a previous study, however, our group demonstrated that these primers amplified nonspecific bands in 28 clinical isolates of 14 species (3). Therefore, we used ITS-F and mycom-2 as a pair of genus-specific primers.
To develop multiplex PCR that will identify clinically important mycobacteria in one reaction tube, the PCR conditions should be the same in the individual reactions. It was most difficult to find conditions under which specific bands were produced successfully under the same PCR conditions, despite variability in the length and GC content of each pair of primers. We studied the influence of TMAC, dimethyl sulfoxide, and glycerol on the PCRs and found that the use of TMAC in the PCR mixture dramatically reduced or eliminated nonspecific priming events, thereby enhancing the specificity of the reaction. In fact, TMAC binds selectively to dA-dT base pairs, altering the dissociation equilibrium and increasing the melting temperature (5). In a solution containing 3.0 M TMAC, this displacement is sufficient to shift the melting temperature of dA-dT base pairs to that of dG-dC base pairs. The PCR yield increased with 15 to 60 µM TMAC, while 150 µM TMAC completely inhibited the reaction (4). In this study, PCR was carried out in the presence of 0, 5, 10, 20, 50, and 100 µM TMAC. We observed an increase in PCR specificity at 10 µM TMAC (data not shown). A nonspecific upper band, of approximately 550 bp, was seen with M. tuberculosis and M. bovis. We did not adjust the concentrations of target DNAs of type strains and clinical isolates. Because we tested PCR with clinical isolates, target DNA was not constant. We optimized the PCR conditions.
In conclusion, the novel primers that we designed could be used to detect and identify mycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitated the early and accurate diagnosis of mycobacteriosis. Further experiments will be necessary to determine the conditions needed to detect mycobacteria directly from patient specimens, such as sputum. At the same time, the conditions for successfully performing multiplex PCR using at least four sets of primers, including genus-specific and M. tuberculosis-, MAC-, and M. fortuitum-specific primers, should be studied.
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ACKNOWLEDGMENT |
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This study was supported by a grant from the Korea Health R & D Project, Ministry of Health & Welfare, Republic of Korea (HMP-99-V-B-0004).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, College of Medicine, Pusan National University, #10, Amidong-1-Ga, Seogu, Pusan 602-739, Korea. Phone: 82-51-240-7725. Fax: 82-51-248-1118. E-mail: kimcm{at}hyowon.cc.pusan.ac.kr.
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Discussion
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Journal of Clinical Microbiology, November 2000, p. 4080-4085, Vol. 38, No. 11
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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