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J Clin Microbiol. 2003 Jun; 41(6): 2605–2615.
PMCID: PMC156504

Detection and Identification of Mycobacterium Species Isolates by DNA Microarray


Rapid identification of Mycobacterium species isolates is necessary for the effective management of tuberculosis. Recently, analysis of DNA gyrase B subunit (gyrB) genes has been identified as a suitable means for the identification of bacterial species. We describe a microarray assay based on gyrB gene sequences that can be used for the identification of Mycobacteria species. Primers specific for a gyrB gene region common to all mycobacteria were synthesized and used for PCR amplification of DNA purified from clinical samples. A set of oligonucleotide probes for specific gyrB gene regions was developed for the identification of 14 Mycobacterium species. Each probe was spotted onto a silylated glass slide with an arrayer and used for hybridization with fluorescently labeled RNA derived from amplified sample DNA to yield a pattern of positive spots. This microarray produced unique hybridization patterns for each species of mycobacteria and could differentiate closely related bacterial species. Moreover, the results corresponded well with those obtained by the conventional culture method for the detection of mycobacteria. We conclude that a gyrB-based microarray can rapidly detect and identify closely related mycobacterial species and may be useful in the diagnosis and effective management of tuberculosis.

Tuberculosis is a disease with worldwide significance (7). Effective treatment of tuberculosis requires the rapid detection and identification of Mycobacterium tuberculosis. Culture of the isolates is the traditional method used to confirm a diagnosis of tuberculosis, but culture is time-consuming because M. tuberculosis isolates can take 4 to 8 weeks to grow in culture. A diagnosis can also be made by biochemical or immunological testing, but this can take even longer. Direct staining and microscopic examination of clinical specimens can produce results more quickly, but this methodology lacks sensitivity and specificity.

On the other hand, the AccuProbe system (Gen-Probe, San Diego, Calif.) has been the “gold standard” among the commercial systems that identify mycobacteria by means of DNA probes. However, hybridization with the AccuProbe system that was commercially available at that time was found to fail with a number of strains displaying the phenotypic features of the species Mycobacterium kansasii (34, 41).

PCR, which permits the amplification of specific DNA sequences and multiplies even a single copy of a given DNA sequence by a factor of 1012 (31), has been applied to various fields of diagnosis and has proved to be a most useful tool for the rapid diagnosis of infectious diseases (13, 20, 28). PCR has been used to analyze various mycobacterial genes for diagnostic purposes, including 16S and 23S rRNA genes, genus- and species-specific fragments in the chromosome (8, 11, 16, 26), genes coding for the 65-kDa heat shock protein (2, 15, 24) and the 38-kDa protein B antigen (38), the dnaJ gene (39), and insertion sequences such as IS6110 (9, 14, 30, 37, 40). 16S rRNA has been reported to be a suitable target for use in PCR amplification assays for the detection of Mycobacterium spp. in a variety of clinical samples (21) and has frequently been used to identify various specific microorganisms because 16S rRNA genes show species-specific polymorphisms (5, 18, 22, 25). However, because of the extremely slow speed of the molecular evolution of 16S rRNA, the number of substituted bases between the 16S rRNA genes of closely related bacterial strains, such as those belonging to the M. tuberculosis complex, is either nonexistent or too small to differentiate between these species.

As an alternative to 16S rRNA analysis, Yamamoto and Harayama (44, 45, 46) designed a set of PCR primers that allowed both the amplification of the gyrB gene, which encodes the subunit B protein of DNA gyrase (topoisomerase type II), and the rapid nucleotide sequencing of the amplified gyrB fragments from a wide variety of bacteria. They used these gyrB genes in the taxonomic classification of Pseudomonas putida and Acinetobacter strains. We have reported that such closely related bacteria, for example, Shigella and Escherichia coli, might be classified by gyrB analysis (12). The rate of molecular evolution inferred from gyrB gene sequences is faster than that inferred from 16S rRNA gene sequences. For detection of Mycobacterium species, Kasai et al. (19) have determined the gyrB gene sequences of 43 slowly growing strains belonging to 15 species in the genus Mycobacterium and developed a method of PCR and PCR-restriction fragment length polymorphism analysis to differentiate these species.

The identification of bacteria by molecular genetics can be advanced further by DNA microarray technology (23, 27, 35). The DNA microarray or DNA chip generally comprises a glass surface on which multiple DNA probes with known identities are fixed for molecular hybridization with DNA samples, which allows the examination of parallel gene expression or genotyping. This method allows the simultaneous analysis of thousands of genes in a short assay time and so is useful for phylogenetic analysis and species identification. For the identification of bacteria, this method may involve the labeling of in vitro RNA transcribed from a target gene from bacteria in specimens, subsequent hybridization of the labeled in vitro transcribed RNA to species-specific oligonucleotide probes on a microarray, and detection of the label, usually by fluorescence. For example, the Affymetrix Genechip, which uses large sets of oligonucleotides that are synthesized rather than spotted onto a glass substrate, has been successfully applied by using 16S rRNA genes as a target for the identification of Mycobacterium species isolates (42).

In the present study, we have investigated the use of a microarray technology with gyrB-derived DNA probes to differentiate Mycobacterium isolates at the species level. Using the nucleotide sequence data in GenBank (Bethesda, Md.), we designed specific probes for the identification of the Mycobacterium species M. tuberculosis, M. bovis, M. africanum, M. avium, M. intracellulare, M. kansasii, M. gordonae, M. asiaticum, M. gastri, M. malmoense, M. marinum, M. scrofulaceum, M. simiae, and M. szulgai. We show that species-specific hybridization patterns on a microarray containing these probes can differentiate and identify these mycobacteria to the species level.


Sample preparation for bacterial strain identification.

The strains used in this study originated from reference collections (the American Type Culture Collection) or were clinical isolates. One or two freshly grown colonies of bacteria were scraped into a 1.5-ml Eppendorf tube and resuspended in 500 μl of sterile water. The bacterial suspension was then boiled (at 100°C for 10 min) to release the DNA.

Sample preparation for clinical application.

Clinical sputum samples were obtained from the Japan Anti-Tuberculosis Association in Fukujuji Hospital. Some standard strains were obtained from the American Type Culture Collection and used as control bacteria. They were processed by the N-acetyl-l-cysteine (NALC)-NaOH method (29) and used for direct identification assays with the microarray or by the AMPLICOR MTB-PCR (Roche Diagnostics, Inc., Tokyo, Japan). An equal volume of the NALC-NaOH solution (2% NaOH, 1.45% sodium citrate, 0.5% NALC) was mixed with the processed samples, and the mixture was incubated at room temperature for 20 min. Phosphate buffer (67 mM; pH 6.8) was added, and the mixture was centrifuged (3,500 × g) for 25 min. The excess fluid was poured off, and the sediment was resuspended in 1.0 ml of phosphate buffer; 0.8 ml of this suspension was used for culture by standard methods (29), and 0.2 ml was used to isolate chromosomal DNA, as follows. The cells (0.2 ml) were added to 2× TES buffer (Tris-HCl [pH 8.5], 20 mM; EDTA, 2 mM; NaCl, 300 mM) containing 100 μg of proteinase K (Roche Diagnostics, Basel, Switzerland) and 10% sodium dodecyl sulfate (SDS; final concentration, 1%), and the mixture was heated at 65°C for 1 h. The lysates were extracted with an equal volume of phenol-chloroform and precipitated with ethanol. The pellet was resuspended in 10 μl of TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA).

Human control DNA was also extracted from peripheral blood leukocytes from healthy volunteers by standard techniques (33).

Preparation of DNA microarray.

The oligonucleotides used to prepare the DNA microarray were synthesized at Sawady Technology Co., Ltd. (Tokyo, Japan). Each 10 μl of the 14- to 15-mer oligonucleotides at a concentration of 200 μM was dispensed into a 96-well microplate with 10 μl of 2× ArrayIt Micro-Spotting solution (TeleChem International, Inc., Sunnyvale, Calif.) per well. The amino acid-modified DNA was printed onto silylated microscope slides with an arrayer (SPBIO 2000; Hitachi Software, Tokyo, Japan). Following printing of the slides, the slides were left at 65°C for 18 h to permit thorough drying of the DNA onto the surface of the silylated slides. After the slides had dried, they were washed in 0.2% SDS at 25°C for 5 min each, twice in distilled H2O (dH2O) at 25°C for 2 min each time, and once in dH2O at 95°C for 2 min; cooled to 25°C for 5 min; washed once in sodium borohydride solution (1.3 g of Na2BH4 dissolved in 375 ml of phosphate-buffered saline and 125 ml of pure ethanol) at 25°C for 5 min, twice in 0.2% SDS for 3 min each time, and twice in dH2O at 25°C for 2 min each time; and then left to air dry.

PCR and in vitro RNA transcription.

The gyrB region was amplified by PCR with Mycobacterium genus-specific primers (nucleotide positions 794 to 818 and 894 to 910 in the reference M. tuberculosis sequence in GenBank [accession number AB014241]; the M. tuberculosis amplicon size is 184 bp). The Mycobacterium species-specific primers were derived from regions of the gyrB gene that are conserved among all mycobacterial species. The primers that we designed were F119 (5′-TGGGCAACACCGAAGTGAAGTCGTT-3′) and R184T7 (5′-GTAATACGACTCACTATAGGGCCGCACCARYTCWCGYGCYTT-3′),which contained a bacteriophage T7 promoter sequence at the 5′ ends. Chromosomal DNAs were amplified by PCR in a thermocycler 480 (Perkin-Elmer Co., Norwalk, Conn.). PCR was performed in a total volume of 100 μl with 5 U of Taq DNA polymerase (AmpliTaq; Perkin-Elmer Co.), 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.001% (wt/vol) gelatin, 200 mM each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), 10 μM primer F119, and 10 μM primer R184T7. A 5-μl bacterial sample was added to the PCR solution, which underwent an initial denaturation step of 95°C for 5 min before 30 cycles of 96°C for 1 min, 63°C for 1 min, and 72°C 1 min and then a final step of 72°C for 7 min for the last cycle. The PCR products were analyzed by electrophoresis on a 3% agarose gel. The promoter-tagged PCR amplicons were used to generate fluorescently labeled single-stranded RNA targets by in vitro transcription. Each 50-μl reaction mixture contained approximately 50 ng of PCR product; 20 U of T7 RNA polymerase (Promega, Madison, Wis.); 40 mM Tris-HCl (pH 8.1); 6 mM MgCl2; 2 mM spermidine; 10 mM NaCl; 10 mM dithiothreitol; 2 mM (each) ATP, CTP, and GTP; 0.04 mM UTP; and 0.2 mM Fluoorlink cyanine 5 (Cy-5)-UTP (Amersham Pharmacia Biotech, Piscataway, N.J.). The reaction was carried out at 37°C for 1 h, and then the template DNA was removed by adding 3 μl of DNase I (GIBCO BRL, Grand Island, N.Y.) at 37°C for 15 min. The RNA transcribed in vitro was fragmented by incubation with 30 mM MgCl2 at 94°C for 30 min (42).

Hybridization control.

A hybridization probe (5′-GATCAGACACTTCAAGGTCTAG-3′) was printed onto silylated microscope slides with an arrayer. A DNA probe (5′-CTAGACCTTGAAGTGTCTGATC-3′) labeled with Fluoorlink Cy-5-CTP (Amersham Pharmacia Biotech), together with labeled sample DNA, was allowed to hybridize to the microarray. The control probe and the complementary target were made such that, ideally, they had similar melting temperatures and did not have consensus sequences that were the same as the sequence of the other probe. The hybridization signals for the control probes were used as hybridization controls.

Hybridization and analysis.

The fluorescently labeled RNA was resuspended in 2.0 μl of sterile water and then in 8.0 μl of prewarmed 1.25× UniHyb hybridization solution (TeleChem International, Inc.). The microarray was incubated in the presence of the fragmented labeled RNA solution for 30 min at 30°C and then washed in 2× SSC (0.3 M NaCl plus 30 mM sodium citrate)-0.02% SDS at 25°C for 3 min and in 0.2× SSC at 25°C for 30 s. The fluorescent signal emitted by a target bound to the microarray was detected at a pixel resolution of 10 μm by using the ScanArray Lite instrument (GSI Lumomics, Northville, Mass.). Sixteen-bit TIFF images of 10-μm resolution were imported into QuantArray software (GSI Lumomics). After subtraction of the background intensity (by a fixed circle-based quantification method), the mean intensities of the individual spots were used to calculate match-to-mismatch signal intensity ratios for pairs of spots corresponding to different alleles.

Effect of target sequence on signal intensity.

To determine whether the oligonucleotide DNA targets arrayed retained their expected hybridization properties, we first tested the hybridization signal intensities on the microarray by comparing the differences in fluorescence intensities between spots encoding homologous targets and those encoding nonhomologous targets. A perfectly matched oligonucleotide probe (M1-1 [5′-ACCGACGCGAAAGT-3′]) and a mismatched oligonucleotide probe (M1-2 [5′-ACCGACTCGAAAGT-3′) (where the underscores indicate the mismatched nucleotide]) were printed onto silylated microscope slides (n = 5) with an arrayer. The Fluoorlink Cy-5-CTP (Amersham Pharmacia Biotech)-labeled DNA probe (5′-ACTTTCGCGTCGGT-3′) was allowed to hybridize to the microarray, and then the hybridization signals were analyzed.

Direct M. tuberculosis amplification tests.

PCRs for M. tuberculosis amplification (MTB-PCR; AMPLICOR) were performed according to the instructions of the manufacturer (1).

Nucleotide sequence accession numbers.

The nucleotide sequence data reported in this paper appear in the GenBank nucleotide sequence database under the following accession numbers: AB014192, AB014206, AB014189, AB014184, AB014294, AB014191, AB014188, AB014302, AB014187, AB014203, AB014205, AB014027, AB014182, AB014185, and AB014242.


Amplification of mycobacterial species.

The DNAs of the mycobacterial species from clinical specimens were amplified and analyzed by 3% agarose gel electrophoresis to confirm that the primers were specific for the gyrB genes of all Mycobacterium species. A representative example of the mycobacterial DNA amplification efficiencies is shown in Fig. Fig.1.1. We obtained specific amplification of a 184-bp DNA fragment by PCR with primers F119 and R184T7. No amplification products were observed from human genomic DNA (Fig. (Fig.1,1, lane 10), indicating that there is no similar or homologous region in human DNA. These results show that only a single band was amplified from the clinical specimens and that the primers used were appropriate for amplification of the gyrB region of mycobacteria at the genus level.

FIG. 1.FIG. 1.FIG. 1.
Amplification of mycobacterial DNAs with primers F119 and R184T7. Lanes: 1 to 8, amplification of gyrB fragments from clinical samples; 1, M. scrofulaceum; 2, M. tuberculosis; 3, M. kansaii; 4, M. intracelluare; 5, M. gordonae; 6, M. avium; 7, M. bovis ...

Effect of target sequence on signal intensity.

A DNA probe (5′-ACTTTCGCGTCGGT-3′) labeled with Fluoorlink Cy-5-CTP (Amersham Pharmacia Biotech) was allowed to hybridize to the microarray, and then the hybridization signals were analyzed. Figure Figure22 shows that the signal intensities varied from 85,000 to 200 fluorescence units. Quantification of the fluorescence signals showed that the relative intensity ratio of the homologous target to the nonhomologous target was about 8.0 for three different concentrations. These data show that these probes can differentiate between signal intensities arising from homologous and nonhomologous targets.

FIG. 2.
Quantification for the hybridization signals. (a) Perfectly matched oligo-probe (PM) (M1-1; 5′ ACCGACGCGAAAGT 3′) and mismatched oligo-probe (MM) (M1-2; 5′ ACCGACTCGAAAGT 3′) were printed onto silylated microscope slides ...

Design of species-specific oligonucleotides for microarray analysis.

The sequence alignment in Fig. Fig.33 was used to identify regions that were both unique to one particular mycobacterial species and sufficiently different from all other species to avoid cross-hybridization (17, 32). In addition, we designed each probe of 15 bases in such a way that the species-specific base sequence was located in the center of the probe. The probes chosen for each mycobacterial species sequenced are shown in Table Table11.

FIG. 3.
Nucleotide sequence alignment of the gyrB genes from the 14 Mycobacterium species strains. Nucleotides identical to those in M. tuberculosis gyrB are indicated with dots, and PCR primers are underlined.
Mycobacteria-specific probes

Because most Mycobacterium species cannot be identified with a single probe, we used a combination of probes to identify individual species. Figure Figure44 indicates how a set of 28 probes can be used to differentiate 14 Mycobacterium species. Each Mycobacterium species was expected to show a unique pattern of reactivity to this set of probes. For example, whereas a sample with M. tuberculosis is expected to hybridize with the M1-1, M3-1, M4-1, and M5-1 probes, no other Mycobacterium species is able to react with the exact same four probes.

FIG. 4.FIG. 4.
Positions of Mycobacterium-specific oligo-probes on the DNA microarray. Each species of Mycobacterium was expected to show a unique pattern of reactivity to this set of probes. (A) M. tuberculosis; (B) M. bovis; (C) M. avium; (D) M. intracellulare; (E) ...

The probes were printed onto the microarray as shown in Fig. Fig.4.4. Figure Figure55 shows the actual scanning image obtained on the microarray, in which the colors of the spots, which are pseudo-colored from yellow (highest) to blue (background level) according to the fluorescence intensity, represent the intensities of the reactions with the probes. Positive spots could be differentiated from negative spots, and a clear, specific pattern of reactivity was observed for each six Mycobacterium species which had been identified by the culture method. The culture results were consistent with the predictions in Fig. Fig.5.5. The specific set of probes complementary to M. tuberculosis, for example, hybridized only to nucleic acids from the corresponding species and not to nucleic acids from any other mycobacterial species examined here.

FIG. 5.
Mycobacterium microarray. Colors represent the various intensities of the Mycobacterium-specific oligonucleotide probes. Microarray analysis was performed after amplification of DNAs from cultured clinical specimens with primers F99 and R184T7, immobilization ...

Microarray assay with labeled RNA from DNA samples from cultured specimens.

We analyzed in a blinded fashion 68 cultured specimens, including specimens with mycobacteria and nonmycobacteria, by both the conventional culture method and the microarray method, and the data were coded at a later time (Table (Table2).2). Both methods gave the same results. The 28 specimens identified as nonmycobacteria by the culture method were classified as negative controls and were not recognized by the microarray, as expected. In the culture assay, five specimens (specimens 6 to 10) were identified at best to be members of the M. avium-M. intracellulare complex (MAC). By contrast, the microarray method was able to identify the individual species in these five specimens, that is, to specify either M. avium or M. intracellulare. For the isolate in one specimen (specimen 15) that was identified as MAC by the culture method, the scanning image obtained on the microarray showed a composite of the pattern specific for M. avium and that specific for M. intracellulare, suggesting that this patient had a dual infection.

Comparison of culture and microarray results with clinical outcome

Blind test of microarray and AMPLICOR assays for direct identification of clinical specimens.

To determine whether the gyrB fragment could be directly detected in clinical samples, we analyzed 122 sputum samples by both the microarray assay and AMPLICOR assays (for M. tuberuclosis, M. avium, and M. intracellulare). Both methods produced identical results for these mycobacteria (Table (Table3).3). Most specimens were both AMPLICOR MTB-PCR and microarray assay negative. Ten of 122 specimens were positive by the microarray assay and the AMPLICOR assay for M. tuberculosis, and 6 and 5 of 122 specimens were positive by both methods for M. avium and M. intracellulare, respectively (Table (Table3).3). Moreover, of six specimens that were positive for M. avium by both methods, the microarray assay identified two cases of dual infections with M. avium-M. intracelluare and M. avium-M. kansasii. Likewise, the microarray method detected a case of dual infection with M. avium-M. intracellulare, for which the AMPLICOR method detected only M. intracelluare.

Comparison of results of microarray analyses with those of the AMPLICOR system


Mycobacterial species are usually identified by time-consuming culture methods. Recently, the development of rapid diagnostic tests that use molecular genetic methods, such as PCR amplification, has been reported. The microarray has proved to be a valuable tool for the specific detection of microorganisms directly from clinical samples. In particular, it has several advantages over classical detection methods: first, the number of organisms in a clinical sample is not always large enough for the organisms to be detected by microscopic methods; second, the period required for culture of these organisms is long; finally, not all acid-fast bacilli are M. tuberculosis, so an identification test must be used to differentiate Mycobacterium species.

Among several DNA regions that have been targeted for diagnostic detection of Mycobacterium species, the 16S rRNA gene has been used the most frequently. DNA detection has been further advanced by exploiting the DNA microarray technology (27), which can simultaneously detect hybridization to multiple DNA probes arranged in an array.

In the study described here we have developed a microarray assay for the detection of Mycobacterium species that uses the gyrB gene as the target. Our study shows that a microarray assay targeting the gyrB gene can identify mycobacteria at the species level and can even differentiate among closely related species. A previous study (42) used 16S rRNA sequence data to construct a DNA probe array for the detection of mycobacteria, but that array could not distinguish among closely related species of mycobacteria. In contrast, our microarray analysis with the gyrB gene was able to classify the closely related species M. tuberculosis and M. bovis. Of possible therapeutic relevance was the fact that closely related species of very different clinical importance were clearly differentiated by this technique, as M. tuberculosis complex species could be distinguished from M. avium, M. marinum, M. asiaticum, and M. intracellulare. These results confirm those of previous studies reported by Yamamoto and Harayama (44, 45, 46) and Kasai et al. (19) that analysis of gyrB gene sequences is a rapid and effective method for the identification of bacterial species.

We have also shown that the microarray can readily differentiate between correctly matched and mismatched sequences (Fig. (Fig.2).2). The hybridization signals arising from perfectly matched oligonucleotide probes and mismatched oligonucleotide probes gave a signal-to-background ratio of 8.0 for three concentrations of arrayed DNA (10−4, 10−5, and 10−6 M).

We compared the clinical performance of the microarray assay with that of the traditional culture method. The results for isolates from 68 clinical specimens, including mycobacteria and nonmycobacteria, show that the overall performance of the microarray was comparable to that of the culture method. In cases in which the culture assay could identify specimens only as members of MAC, however, the microarray method was able to identify the individual species, that is, either M. avium or M. intracellulare. The microarray method was also able to identify both types of bacteria in cases of dual infection. For example, one specimen identified as MAC by the culture method was identified to include both M. avium and M. intracellulare through its composite pattern on the microarray (Table (Table3).3). Our assay also performed well when its performance was compared with that of the AMPLICOR MTB-PCR assay. Studies on the use of PCR for the detection of M. tuberculosis organisms show that overall it has good sensitivity and specificity, although the results for sensitivity vary from approximately 50 to 100% (3, 4, 6, 10, 24, 36, 43). Cartuyvels et al. (4) have reported that the AMPLICOR PCR cannot yet replace culture as a first-line screening method for the detection of M. tuberculosis isolates, but it can be used as a rapid confirmatory test for smear-positive specimens or in the case of a strongly suspected M. tuberculosis infection (36). Rapid identification of Mycobacterium species is an important factor for the successful diagnosis of mycobacteriosis. However, because of the variable nature of the sputum specimens and the low sensitivities of these tests, there is a risk of false-negative results. No false-negative results were obtained by the microarray assay in this study.

Our assay offers several advantages over other assays described in the literature. In addition, these initial studies suggest that our microarray method is at least as sensitive as and may be less subject to error than methods based only on PCR. Most importantly, the microarray assay can analyze a sample for several kinds of bacteria at the same time. Dual infections, such as those caused by M. avium and M. intracellulare or M. avium and M. kansasii, could also be identified by microarray analysis. Thus, we have shown that the microarray assay described here has high levels of analytical sensitivity and specificity as a clinical test.

In summary, the potential of the microarray strategy for the parallel testing of different targets has been demonstrated. It has also been shown that gyrB gene-based microarrays have the potential to be used for the direct testing of samples to provide rapid results for species identification. Future studies will focus on defining both the identification of bacterial species and the drug resistance genotyping features of this technology for application in clinical diagnostics.


We thank Kazunori Hochido for assistance with culture. We thank Noboru Fujinami and Yumiko Saito for stimulating discussions. We also thank Tadashi Matsunaga of the Tokyo University of Agriculture and Technology for critical advice regarding this study.

This study was supported by The New Energy and Industrial Technology Development Organization of Japan.


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