This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wada, T.
Right arrow Articles by Kobayashi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wada, T.
Right arrow Articles by Kobayashi, K.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, November 2004, p. 5277-5285, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5277-5285.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Dual-Probe Assay for Rapid Detection of Drug-Resistant Mycobacterium tuberculosis by Real-Time PCR

Takayuki Wada,1 Shinji Maeda,2* Aki Tamaru,3 Shigeyoshi Imai,4 Atsushi Hase,1 and Kazuo Kobayashi2

Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences,1 Department of Host Defense, Osaka City University Graduate School of Medicine,2 Department of Microbiology, Osaka Prefectural Institute of Public Health,3 Central Clinical Laboratory, Osaka City University Hospital, Osaka, Japan4

Received 24 March 2004/ Returned for modification 9 May 2004/ Accepted 11 July 2004


arrow
ABSTRACT
 
Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.


arrow
INTRODUCTION
 
Tuberculosis (TB) presents a significant health threat to the world's population, with 8 million new cases of disease and 2 million deaths per year (36). To minimize the emergence and spread of drug-resistant TB, the basic principle of anti-TB treatment is to administer multiple drugs to which the organism is susceptible. Strains of Mycobacterium tuberculosis that are resistant to anti-TB drugs are being encountered with increased frequency (7). The major risk factors for drug resistance include inadequate prescription and delivery of chemotherapy, poor compliance, and an insufficient number of active drugs in the treatment regimen. The emergence of drug-resistant strains threatens our capability to control TB (2). Multidrug-resistant (MDR) M. tuberculosis, defined as simultaneous resistance to at least isoniazid (INH) and rifampin (RIF), is a serious problem. Strains of MDR M. tuberculosis appear to result from the stepwise acquisition of mutations in the genes encoding drug targets or drug-converting enzymes (10).

Rapid detection of susceptibility or resistance is crucial for TB treatment, because the initial choice of effective drugs is important. Major anti-TB drugs include INH, RIF, and ethambutol (EMB). Genetic studies have demonstrated that more than 95% of RIF resistance is associated with a mutation in the 81-bp core region of the rpoB gene (26, 31). RIF-susceptible mycobacteria do not possess the known mutations in core region of the rpoB gene. Therefore, mutations in the rpoB gene indicate that bacteria are RIF resistant. By contrast, INH-resistant strains exhibit mutations in several genes, such as katG, inhA, oxyR, and ahpC (22, 27, 30). The mutation of codon 315 (Ser) in the catalase-peroxidase (katG) gene is the most frequent site (30 to 65% of resistant strains) (4, 11). Furthermore, EMB-resistant strains have a point mutation at codon 306 (Met) in embB (3, 28), and the frequency is about 70% (3). Mutations of these sites are merely one mechanism leading to drug resistance. Many clinical isolates of drug-resistant M. tuberculosis do not bear mutations in these sites. Even if all of these mutations are examined, some resistant bacteria remain undetectable. However, the presence of mutations in the sites indicates that these bacilli are drug resistant (sensitivity, <100%; positive predictive value, 100%). In drug susceptibility testing, the culture method remains the "gold standard" (15). Since culture requires at least 2 weeks to obtain results (20), rapid diagnosis of drug resistance provides new opportunities for chemotherapeutic intervention in TB.

Because TaqMan minor groove binder (MGB) probes can distinguish one-base mismatches, the real-time PCR system in combination with MGB probes has been applied to analyze single-nucleotide polymorphisms (1, 12). It has been proven that the specificity of MGB probes is quite high (18). In the present study, we have developed a real-time PCR-based system with TaqMan MGB probes to detect the mutations associated with resistance of M. tuberculosis to INH, RIF, and EMB.


arrow
MATERIALS AND METHODS
 
Bacterial strains. To confirm the specificity of the real-time PCR system, we used DNAs extracted from M. tuberculosis H37Rv (ATCC 25618), Mycobacterium bovis (Ravenel), M. bovis BCG (Tokyo), Mycobacterium africanum (ATCC 25420), Mycobacterium microti (TC 77), Mycobacterium avium (ATCC 15769), Mycobacterium intracellulare (ATCC 13950), Mycobacterium kansasii (ATCC 12478), Mycobacterium marinum (ATCC 927), Mycobacterium simiae (ATCC 25275), Mycobacterium asiaticum (ATCC 25276), Mycobacterium xenopi (ATCC 19250), Mycobacterium scrofulaceum (ATCC 19981), Mycobacterium gordonae (ATCC 14470), Mycobacterium malmoense (ATCC 29571), Mycobacterium shimoidei (ATCC 27962), Mycobacterium nonchromogenicum (ATCC 19530), Mycobacterium fortuitum (ATCC 6841), Mycobacterium abscessus (ATCC 19977), Mycobacterium tokaiense (ATCC 27282), Mycobacterium austroafricanum (ATCC 33464), Mycobacterium pulveris (ATCC 35154), Mycobacterium smegmatis (ATCC 14468), and Mycobacterium leprae (Thai 53). M. leprae was a kind gift from M. Matsuoka, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan. In addition to mycobacteria, DNAs from Klebsiella pneumoniae (clinical isolate), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus aureus (ATCC 29213) were used.

Drug-resistant clinical isolates and DNA isolation. Using the proportion method with Ogawa egg medium, drug resistance was defined as growth of at least 1% of the number of colonies that grew on drug-free medium at critical concentrations of the drugs (i.e., 40 mg of RIF per liter, 0.2 mg of INH per liter, and 2 mg of EMB per liter) (35). The present study examined 45 laboratory strains of RIF-resistant M. tuberculosis (resistant to RIF alone, 7 strains; resistant to RIF and INH, 12 strains; resistant to RIF and EMB, 9 strains; resistant to RIF, INH, and EMB, 17 strains). Genomic DNAs of mycobacteria were isolated from bacteria grown on Ogawa medium by combined chloroform extraction and mechanical disruption (16). DNAs from K. pneumoniae, P. aeruginosa, and S. aureus were extracted with a QIAamp DNA Mini kit (Qiagen Inc., Valencia, Calif.). DNA concentration was estimated by UV absorbance at 260 nm.

Clinical samples and DNA preparation. We examined 27 clinical samples of sputum from patients with pulmonary TB. Drug susceptibility testing was performed as described in a previous study (35). A concentrated smear was prepared from specimens that were decontaminated by using the N-acetyl-L-cysteine-NaOH protocol (17). The basic fuchsin (Ziehl-Neelsen) staining procedure was employed. The DNAs were extracted by using the AMPLICOR respiratory specimen preparation kit (Roche Diagnostic Systems, Inc., Branchburg, N.J.) according to the manufacturer's instructions and were confirmed as M. tuberculosis complex with the COBAS AMPLICOR M. tuberculosis detection kit (Roche Diagnostic Systems, Inc.) (25).

PCR amplification and DNA sequencing. DNA sequences of rpoB (GenBank accession no. L27989), embB (U68480), and katG (X68081) were used for primer design. PCR was performed with each primer set (Table 1) as described previously (19). Both strands of PCR products of the rpoB and the embB genes were sequenced by using respective primers that were used in PCR. For DNA sequencing of the katG mutation sites, alternative sequencing primers were designed, because the amplicon (1,771 bp) was much longer than other genes (Table 1). Direct sequencing of PCR products was performed with a CEQ2000 automate sequencer (Beckman Coulter, Inc., Fullerton, Calif.) and a DTCS quick-start master mix kit (Beckman Coulter, Inc.).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Designed primers and probes used in this study

Preparation of TaqMan MGB probes. The TaqMan MGB probes were designed to hybridize with wild-type DNA by using the Primer Express program (Applied Biosystems, Foster City, Calif.). The MGB probes were synthesized by Applied Biosystems. The primers and probes used in the present study are shown in Table 1. Four of eight probes were labeled with 6-carboxyfluorescein (FAM) (emission wavelength, 518 nm) and the remaining four probes were labeled with VIC (emission wavelength, 552 nm), because these two dyes emit luminescence of different wavelengths and can be distinguished individually in one tube.

Real-time PCR and nested PCR. The real-time PCR mixture was prepared in a final volume of 25 µl with 12.5 µl of Universal PCR Master Mix (Applied Biosystems), 25 pmol of primer, the optimal concentration of FAM- and VIC-labeled TaqMan MGB probes (Table 1), and 5 ng of purified DNA or 1 µl of extracted DNA. Real-time PCR analysis was performed with an ABI PRISM 7700 instrument (Applied Biosystems). The conditions of PCR amplification were 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed with Sequence Detector software (Applied Biosystems). Fluorescence of hybridized probes was expressed as {Delta}Rn (normalized reporter signal). The number of amplification cycles required for emission of a certain luminescence intensity by each probe ({Delta}Rn = 0.2) reflected the amount of DNA in the sample. This cycle number was called the threshold cycle (Ct). Therefore, the presence of a mutation would result in an increase in Ct.

Nested PCR was performed when signals were undetected in real-time PCR. The PCR was carried out in a 25-µl reaction volume. The reaction mixture contained 1x PCR buffer, 1 U of GC-rich enzyme mix (Roche Diagnostics Corp., Indianapolis, Ind.), a 200 µM concentration of each of the four deoxynucleoside triphosphates, a 1 µM concentration of three kinds of primer sets (Table 1), 1 M GC-rich solution, 1.5 mM MgCl2, and 10 µl of template DNA. The primer pairs for the rpoB, katG, and embB genes amplified 534-, 464-, and 408-bp fragments, respectively. The PCR conditions were as follows: initial denaturation at 94°C for 5 min and then 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. After the first PCR, the amplified product was diluted 100-fold with sterilized water. One microliter of this solution was used for the real-time PCR analysis as described above.

IPC for detecting PCR inhibitors. For analysis of sputum samples from TB patients, an internal process control (IPC) was performed to detect PCR inhibitors PCR simultaneously. As IPC primers, each rpoB primer was added to the 5' end of the corresponding lambda phage primer (Table 1). The identical sequence of the lambda phage IPC-R probe, which was 5' labeled with FAM and quenched with 6-carboxytetramethylrhodamine (TAMRA) at the 3' end, was used for analysis as described previously (13).

Electrophoresis. Amplification products were separated on 3% agarose gels with 1x Tris-acetate-EDTA buffer for 45 min at 100 V.


arrow
RESULTS
 
Real-time PCR. For rapid detection of mutations in the rpoB, katG, and embB genes involving M. tuberculosis resistance to RIF, INH, and EMB, respectively, three primer pairs and eight MGB probes were designed (Fig. 1 and Table 1). Five probes (rpo510/514, rpo514/520, rpo520/524, rpo524/529, and rpo529/533) were used for detection of mutations in the hot spot of rpoB (81 bp between codons 507 and 533 [equivalent to Escherichia coli numbering system {31}]). One probe (TB control probe) was designed outside the hot spot in rpoB as a control for determining the amount of DNA and for identifying M. tuberculosis. Polymorphisms in the 81-bp region of rpoB could be analyzed by using three tubes. One probe each for embB (codon 306) and katG (codon 315) was labeled with FAM and VIC, respectively. These probes were mixed with their four corresponding primers in one tube. Four tubes (three tubes for control and RIF resistance and one tube for INH and EMB resistance) were employed in the assay. Luminescence of all eight probes was detectable by real-time PCR with genomic DNA extracted from M. tuberculosis H37Rv as the template (Fig. 2A). When the DNA had mutations in rpoB at position 516 and in katG at position 315, the corresponding probes showed no luminescence signal (Fig. 2B). Typical results are shown in Fig. 2C and D. These results indicate that the probes used in this study can identify mutations of the target genes. Similar results were obtained by using autoclaved supernatants of M. tuberculosis suspensions instead of purified DNAs (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1. Design of TaqMan MGB probes for detection of mutations in the rpoB, embB, and katG genes. MGB probes were labeled with two different dyes (FAM or VIC). The DNA sample and PCR primers for rpoB amplification were mixed with each set of probes in three different tubes: (i) TB control and rpo520/524, (ii) rpo510/514 and rpo514/520, and (iii) rpo524/529 and rpo529/533 (A). The emb306 and kat315 probes and embB and katG PCR primers were mixed and reacted in another tube (B). The sequences of primers and probes are listed in Table 1.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. Analysis of DNAs from mycobacteria with eight TaqMan MGB probes by real-time PCR. The templates were genomic DNAs extracted from M. tuberculosis H37Rv (A) as a control, certain mutants of M. tuberculosis (B, rpoB and katG; C, rpoB and embB; and D, rpoB), and M. avium (E). The x axis shows cycle numbers of PCR, and the y axis represents the normalized reporter signal ({Delta}Rn). A horizontal line indicates the threshold ({Delta}Rn = 0.2). The Ct is expressed as the number of cycles to reach the threshold.

Specificity and sensitivity. No luminescence was found when M. avium DNA (up to 50 ng) was analyzed in this system (Fig. 2E). In addition, luminescence was not detected even when more than 50 ng of DNA from other mycobacteria, such as M. intracellulare, M. kansasii, M. marinum, M. simiae, M. asiaticum, M. xenopi, M. scrofulaceum, M. gordonae, M. malmoense, M. shimoidei, M. nonchromogenicum, M. fortuitum, M. abscessus, M. tokaiense, M. austroafricanum, M. pulveris, M. smegmatis, and M. leprae, was used as the template. The presence of sufficient amounts of DNA in the system was confirmed by alternative PCR with mycobacterial 16S rRNA gene primers (6) or dnaJ gene primers (29) (data not shown). As expected, we detected no luminescence with DNAs prepared from K. pneumoniae, P. aeruginosa, and S. aureus. By contrast, the other members of M. tuberculosis complex, such as M. bovis, M. bovis BCG, M. africanum, and M. microti, exhibited similar luminescence in the real-time PCR system (data not shown). Therefore, the specificity of this system for the M. tuberculosis complex was sufficiently high.

By using purified genomic DNA from M. tuberculosis H37Rv, the sensitivity of this system was determined. In direct real-time PCR with the rpoB primer set and TB control probe, 100 fg of genomic DNA could be detected. Therefore, the mutation was detectable efficiently in the presence of 250 fg (Ct = 37) of TB genomic DNA. The detection limit of the real-time PCR was 10 fg of DNA when combined with nested PCR (data not shown).

Assessment of real-time PCR. We extracted and sequenced genomic DNAs from 45 laboratory strains of RIF-resistant M. tuberculosis. They were classified into 20 groups based on their genotypes by nucleotide sequencing of M. tuberculosis DNAs (Table 2). The luminescence intensity of TaqMan MGB probes in the real-time PCR amplification system was expressed as Ct. The Ct was higher when mutations were present in the genes. Using 45 RIF-resistant strains, we measured the Ct derived from an internal TB control probe bound outside the hot spots of the rpoB gene and {Delta}Ct, which expressed the difference between the control and each MGB probe (Table 2). It was found that the {Delta}Cts of probes hybridizing with the region without mutations were low (from –2.44 to 1.61) and that the {Delta}Ct was higher (≥7) when mutations existed in the target DNA that should hybridize with the MGB probe. These results suggest that a {Delta}Ct of more than 7 was associated with a mutation in the nucleotide sequence. No strains that had mutations within the codons 510 to 514 of the rpoB gene were found. To check to the specificity of the rpo510/514 probe, the rpoB gene was cloned and mutations were constructed at codons 511 (CTG->CCG) and 513 (CAA->CTA). Real-time PCR analysis with each construct as the template showed that the luminescence that was derived from probe rpo510/514 disappeared completely (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Luminescent patterns of real-time PCR with DNAs from 45 laboratory strains of RIF-resistant M. tuberculosisa

Clinical application of real-time PCR. DNAs extracted from sputa of patients with pulmonary TB were examined to assess to possibility of rapid detection of drug-resistant M. tuberculosis in clinical specimens by real-time PCR. Twenty-five clinical samples were culture positive, and two samples were culture negative. Nine samples were smear positive, and 18 samples were negative (Table 3). Real-time PCR was used to analyze sputum samples for detection of mutations. Sample C5 had a mutation at codon 315 in the katG gene, and sample C11 possessed mutations at codon 531 in the rpoB gene, codon 315 in the katG gene, and codon 306 in the embB gene. These findings were in agreement with the results obtained by DNA sequencing. The drug susceptibility phenotypes of clinical isolates assessed by conventional culture methods were consistent with the genotypes (Table 3). Thus, our real-time PCR system can detect mutations even when clinical samples, such as sputa, are used.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Luminescence patterns of real-time PCR with DNA extracted from sputua of TB patientsa

A total of 16 of 27 sputum samples (59.3%) showed strong luminescence in real-time PCR. By contrast, 11 samples showed no luminescence even after 40 cycles of PCR amplification. This was probably due to low concentrations, because IPC was positive for all 11 samples. Consequently, we performed site-specific nested PCR when the amount of DNA was small. The rpoB, katG, and embB genes were amplified by PCR with their corresponding primer sets (Fig. 3). By optimizing the PCR conditions with six primers, three fragments that contained the target sites at a similar concentration were amplified (Fig. 3, lane 4). The targets (rpoB, katG, and embB) were amplified by nested PCR with one tube. The targets were then analyzed by using real-time PCR. Nested PCR products could be analyzed for the presence of mutations associated with drug resistance, although DNA was undetectable in a single-step real-time PCR in these 11 samples. When nested PCR was used before real-time PCR, analysis of all 27 samples could be performed appropriately (Table 4). However, in analysis of clinical samples, the Ct was more variable, resulting in a larger {Delta}Ct (from –3.40 to 3.15) than for purified DNAs extracted from laboratory strains (from –2.44 to 1.61).



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 3. DNA analysis of amplicons by simplex and multiplex PCRs. DNA from sputum sample C1 was amplified by using primers for the rpoB, embB, and katG genes. The product sizes of their amplicons were 534, 408, and 464 bp, respectively (lanes 1 to 3, respectively). Three kinds of primer sets for the rpoB, embB, and katG genes were mixed, and multiplex PCR was performed (lane 4). The M lane was a DNA molecular size marker which contained the 100-bp ladder.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Detection of genotypes by real-time PCR


arrow
DISCUSSION
 
The TaqMan MGB probes are currently used for detection of single-nucleotide polymorphisms, because they can distinguish one-base mismatches (1, 12, 18). In the present study, we have attempted to apply these probes to detect drug resistance on a genetic basis. The implication of all studies on the genetic basis of antimicrobial resistance in M. tuberculosis is that the MDR phenotype (defined as simultaneous resistance to at least INH and RIF) is the result of accumulative mutations (10, 23). The major anti-TB drugs are INH, RIF, and EMB. Among them, INH and RIF are the most potent agents. More than 90% of RIF-resistant M. tuberculosis isolates possess a point mutation at the hot spot in the 81-bp region of rpoB (3, 26). For that reason, detection of mutations in the rpoB gene is a quite useful strategy for diagnosis.

The present study also analyzed extracted DNAs from M. tuberculosis by real-time PCR for the presence of mutations in the katG (codon 315) and embB (codon 306) genes. Codons 315 in katG and 306 in embB were selected because mutations at these sites have been observed frequently in INH- and EMB-resistant M. tuberculosis (3, 4, 11, 28). However, the role of codon 306 in the embB gene for EMB resistance remains controversial, because the mutation has also been found in EMB-susceptible M. tuberculosis. It has been reported that embB306 mutations were detected in 48% of EMB-resistant strains and in 31% of EMB-susceptible strains (21). In particular, 60% of EMB-susceptible strains were resistant to rifampin and isoniazid (i.e., MDR), but none of pansusceptible strains harbored an embB306 mutation. A discrepancy between the results of phenotypic and genotypic analyses of EMB resistance tests was restricted to the strains that were already resistant to other anti-TB drugs, such as MDR M. tuberculosis. Our results have shown that 42% of EMB-susceptible strains of MDR M. tuberculosis had the mutation, in contrast to 0% of pansusceptible strains. When a mutation exists at embB306 in MDR M. tuberculosis, it is possible that the strain is susceptible to EMB. Nevertheless, 70% of EMB-resistant strains have a point mutation at codon 306 in the embB gene (3, 28). The mutation of codon 315 (Ser) in the katG gene is the most frequently encountered mutation that is associated with INH resistance (30 to 65%) (4, 11). In the present study, we found that 70% of INH-resistant strains and 6% of INH-susceptible strains had the mutation at this site. These results are consistent with previous reports (4, 11, 21).

The results obtained from the real-time PCR system in this study were consistent with DNA sequencing analyses of 45 laboratory strains and 27 clinical samples. The system did not react with DNAs prepared from K. pneumoniae, P. aeruginosa, S. aureus, and nontuberculous mycobacteria but reacted solely with DNAs from isolates belonging to the M. tuberculosis complex. These results imply that the primers and probes used here are specific for M. tuberculosis complex.

Although understanding the mechanisms of drug resistance has practical implications for rapid detection of drug-resistant TB by molecular methods, there are a number of limitations to widespread use of PCR-based techniques. For one, resistance to anti-TB agents involves changes in multiple genes and at multiple possible locations within a gene. This fact complicates testing for the various genes. In addition, not all possible genes or mechanisms of resistance have been identified, which represents a significant drawback for diagnostics.

It has been reported previously that real-time PCR with MGB probes was applied for detection of INH-resistant M. tuberculosis (34). In that study, DNAs prepared from smear-positive sputa were used. However, the results for certain samples were inconsistent with those of DNA sequencing. One possible explanation for this is that the amount and/or quality of DNA was not standardized in the study (34). Our results suggest that the amount of DNA in the sample is critical for analysis with real-time PCR using TaqMan MGB probes. The quality of DNA (e.g., whether it is inhibitor free) is also important. In the present study, two control probes were designed for the real-time PCR. One was a TB control probe for identification of M. tuberculosis and confirmation of DNA amount; the other was the IPC for detection of PCR inhibitors. If luminescence is not detected, the reason mentioned above can be suspected.

Several molecular methods to detect drug-resistant M. tuberculosis have been reported (8). These methods are fundamentally based on the detection of point mutations. PCR-DNA sequencing is a straightforward technology to detect mutation (14), although it takes 1 to 2 days until the result is obtained. Methods based on real-time PCR have utilized fluorescence resonance energy transfer (FRET) probes (9, 32), molecular beacons (6, 23, 24), and TaqMan MGB probes (34). By using the real-time PCR as described here, the time to obtain results for drug susceptibility or resistance can be shortened to as little as 3 h from the preparation of DNAs from isolates of M. tuberculosis and sputum samples. Rapid detection of drug resistance or susceptibility may open new therapeutic avenues for intervention in diseases in which drug resistance is a major impediment to treatment. The method based on FRET probes requires a longer probe, because it utilizes both sensor and anchor probes (33). The shift of melting temperature was unclear in some cases when long probes were used in FRET analysis to detect mutations. By contrast, molecular beacons and TaqMan MGB probes, which can be designed to be shorter than FRET probe, detect the mutations on the basis of either emission or lack of emission of luminescence. The luminescence can be measured with a fluorescence plate reader after conventional PCR, even without a real-time PCR analyzer (5).

Molecular beacons are an alternative approach to detect drug-resistant organisms in a real-time format. It has been reported that RIF-resistant M. tuberculosis can be detected in a one-tube reaction by using the beacons (6). This is a convenient and simple method to detect mutations. However, it is necessary to use a real-time PCR analyzer that can detect five kinds of different luminescence simultaneously. Although the assay described here requires the use of four tubes per sample, only two wavelengths are employed, enabling the use of less advanced equipment.

Our present study suggests that drug-resistant M. tuberculosis can be detected by {Delta}Ct with TaqMan MGB probes in real-time PCR. Based on the result for 45 laboratory strains and 27 clinical samples of M. tuberculosis, {Delta}Ct was less than 3.5 when organisms had no mutation (Tables 2 and 3). When {Delta}Ct was more than 6, the DNA samples contained mutations within the target region. These isolates or samples are strongly inferred to be drug-resistant M. tuberculosis (Tables 2 and 3). When wild-type and mutated DNAs were mixed at ratios ranging from 8:2 to 2:8, {Delta}Ct was distributed between 3.5 and 6.0 (data not shown). Indeed, {Delta}Ct of a mixture of drug-susceptible and -resistant bacilli ranged from 3.5 to 6.0. When {Delta}Ct is distributed within the range of 3.5 to 6.0, DNA sequencing analysis should be performed to confirm the mixture. In analysis of clinical samples without mutations, the range of {Delta}Ct became larger (–3.40 to 3.15) than that of purified DNAs extracted from laboratory strains (–2.44 to 1.61). It is thought that {Delta}Ct obtained from the real-time PCR is a useful marker for discriminating between drug-susceptible and drug-resistant M. tuberculosis strains. These criteria are applicable to detect drug-resistant M. tuberculosis in clinical laboratories.

Real-time-based PCR shows sufficient specificity and sensitivity to detect drug-resistant M. tuberculosis even with sputum samples from TB patients without culture. The real-time PCR described here can detect drug-resistant M. tuberculosis within 3 h from DNA preparation. For those reasons, it may be a powerful tool for control of drug-resistant M. tuberculosis.


arrow
ACKNOWLEDGMENTS
 
This work was supported by grants from the Ministry of Health, Labour and Welfare (Research on Emerging and Re-emerging Infectious Diseases, Health Sciences Research Grants); the Ministry of Education, Culture, Sports, Science and Technology; the Ministry of the Environment (Global Environment Research Fund); Osaka City University (Urban Research Project); and the United States-Japan Cooperative Medical Science Program against Tuberculosis and Leprosy.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Host Defense, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. Phone: 81-6-6645-3746. Fax: 81-6-6645-3747. E-mail: smaeda{at}med.osaka-cu.ac.jp. Back


arrow
REFERENCES
 
    1
  1. An, P., G. W. Nelson, L. Wang, S. Donfield, J. J. Goedert, J. Phair, D. Vlahov, S. Buchbinder, W. L. Farrar, W. Modi, S. J. O'Brien, and C. A. Winkler. 2002. Modulating influence on HIV/AIDS by interacting RANTES gene variants. Proc. Natl. Acad. Sci. USA 99:10002-10007.[Abstract/Free Full Text]
    2. 2
  2. Bloom, B. R. 2002. Tuberculosis—the global view. N. Engl. J. Med. 346:1434-1435.[Free Full Text]
    3. 3
  3. Cockerill, F. R., III. 1999. Genetic methods for assessing antimicrobial resistance. Antimicrob. Agents Chemother. 43:199-212.[Free Full Text]
    4. 4
  4. Cockerill, F. R., III, J. R. Uhl, Z. Temesgen, Y. Zhang, L. Stockman, G. D. Roberts, D. L. Williams, and B. C. Kline. 1995. Rapid identification of a point mutation of the Mycobacterium tuberculosis catalase-peroxidase (katG) gene associated with isoniazid resistance. J. Infect. Dis. 171:240-245.[Medline]
    5. 5
  5. de Kok, J. B., E. T. Wiegerinck, B. A. Giesendorf, and D. W. Swinkels. 2002. Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes). Hum. Mutat. 19:554-559.[CrossRef][Medline]
    6. 6
  6. El-Hajj, H. H., S. A. Marras, S. Tyagi, F. R. Kramer, and D. Alland. 2001. Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons. J. Clin. Microbiol. 39:4131-4137.[Abstract/Free Full Text]
    7. 7
  7. Espinal, M. A., A. Laszlo, L. Simonsen, F. Boulahbal, S. J. Kim, A. Reniero, S. Hoffner, H. L. Rieder, N. Binkin, C. Dye, R. Williams, M. C. Raviglione, et al. 2001. Global trends in resistance to antituberculosis drugs. N. Engl. J. Med. 344:1294-1303.[Abstract/Free Full Text]
    8. 8
  8. Garcia de Viedma, D. 2003. Rapid detection of resistance in Mycobacterium tuberculosis: a review discussing molecular approaches. Clin. Microbiol. Infect. 9:349-359.[CrossRef][Medline]
    9. 9
  9. Garcia de Viedma, D., M. del Sol Diaz Infantes, F. Lasala, F. Chaves, L. Alcala, and E. Bouza. 2002. New real-time PCR able to detect in a single tube multiple rifampin resistance mutations and high-level isoniazid resistance mutations in Mycobacterium tuberculosis. J. Clin. Microbiol. 40:988-995.[Abstract/Free Full Text]
    10. 10
  10. Gillespie, S. H. 2002. Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob. Agents Chemother. 46:267-274.[Free Full Text]
    11. 11
  11. Heym, B., P. M. Alzari, N. Honore, and S. T. Cole. 1995. Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis. Mol. Microbiol. 15:235-245.[Medline]
    12. 12
  12. Holloway, J. W., B. Beghe, S. Turner, L. J. Hinks, I. N. Day, and W. M. Howell. 1999. Comparison of three methods for single nucleotide polymorphism typing for DNA bank studies: sequence-specific oligonucleotide probe hybridisation, TaqMan liquid phase hybridisation, and microplate array diagonal gel electrophoresis (MADGE). Hum. Mutat. 14:340-347.[CrossRef][Medline]
    13. 13
  13. Jensen, J. S., E. Bjornelius, B. Dohn, and P. Lidbrink. 2004. Use of TaqMan 5' nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic. J. Clin. Microbiol. 42:683-692.[Abstract/Free Full Text]
    14. 14
  14. Kapur, V., L. L. Li, S. Iordanescu, M. R. Hamrick, A. Wanger, B. N. Kreiswirth, and J. M. Musser. 1994. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J. Clin. Microbiol. 32:1095-1098.[Abstract/Free Full Text]
    15. 15
  15. Kent, P. R., and G. P. Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. U.S. Department of Health and Human Services, Washington, D.C.
    16. 16
  16. Kozak, M. 1987. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15:8125-8148.[Abstract/Free Full Text]
    17. 17
  17. Kubica, G. P., W. E. Dye, M. L. Cohn, and G. Middlebrook. 1963. Sputum digestion and decontamination with N-acetyl-L-cysteine-sodium hydroxide for culture of mycobacteria. Am. Rev. Respir. Dis. 87:775-779.[Medline]
    18. 18
  18. Kutyavin, I. V., I. A. Afonina, A. Mills, V. V. Gorn, E. A. Lukhtanov, E. S. Belousov, M. J. Singer, D. K. Walburger, S. G. Lokhov, A. A. Gall, R. Dempcy, M. W. Reed, R. B. Meyer, and J. Hedgpeth. 2000. 3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 28:655-661.[Abstract/Free Full Text]
    19. 19
  19. Maeda, S., M. Matsuoka, N. Nakata, M. Kai, Y. Maeda, K. Hashimoto, H. Kimura, K. Kobayashi, and Y. Kashiwabara. 2001. Multidrug resistant Mycobacterium leprae from patients with leprosy. Antimicrob. Agents Chemother. 45:3635-3639.[Abstract/Free Full Text]
    20. 20
  20. Martin-Casabona, N., D. Xairo Mimo, T. Gonzalez, J. Rossello, and L. Arcalis. 1997. Rapid method for testing susceptibility of Mycobacterium tuberculosis by using DNA probes. J. Clin. Microbiol. 35:2521-2525.[Abstract]
    21. 21
  21. Mokrousov, I., T. Otten, B. Vyshnevskiy, and O. Narvskaya. 2002. Detection of embB306 mutations in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis from northwestern Russia: implications for genotypic resistance testing. J. Clin. Microbiol. 40:3810-3813.[Abstract/Free Full Text]
    22. 22
  22. Morris, S., G. H. Bai, P. Suffys, L. Portillo-Gomez, M. Fairchok, D. Rouse, A. Telenti, N. Honore, C. Bernasconi, J. March, A. Ortega, B. Heym, H. E. Takiff, and S. T. Cole. 1995. Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis. J. Infect. Dis. 171:954-960.[Medline]
    23. 23
  23. Piatek, A. S., A. Telenti, M. R. Murray, H. El-Hajj, W. R. Jacobs, Jr., F. R. Kramer, and D. Alland. 2000. Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing. Antimicrob. Agents Chemother. 44:103-110.[Abstract/Free Full Text]
    24. 24
  24. Piatek, A. S., S. Tyagi, A. C. Pol, A. Telenti, L. P. Miller, F. R. Kramer, and D. Alland. 1998. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat. Biotechnol. 16:359-363.[CrossRef][Medline]
    25. 25
  25. Rajalahti, I., P. Vuorinen, M. M. Nieminen, and A. Miettinen. 1998. Detection of Mycobacterium tuberculosis complex in sputum specimens by the automated Roche Cobas Amplicor Mycobacterium Tuberculosis Test. J. Clin. Microbiol. 36:975-978.[Abstract/Free Full Text]
    26. 26
  26. Ramaswamy, S., and J. M. Musser. 1998. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber. Lung Dis. 79:3-29.[CrossRef][Medline]
    27. 27
  27. Sreevatsan, S., X. Pan, Y. Zhang, V. Deretic, and J. M. Musser. 1997. Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities. Antimicrob. Agents Chemother. 41:600-606.[Abstract]
    28. 28
  28. Sreevatsan, S., K. E. Stockbauer, X. Pan, B. N. Kreiswirth, S. L. Moghazeh, W. R. Jacobs, Jr., A. Telenti, and J. M. Musser. 1997. Ethambutol resistance in Mycobacterium tuberculosis: critical role of embB mutations. Antimicrob. Agents Chemother. 41:1677-1681.[Abstract]
    29. 29
  29. Takewaki, S., K. Okuzumi, H. Ishiko, K. Nakahara, A. Ohkubo, and R. Nagai. 1993. Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes. J. Clin. Microbiol. 31:446-450.[Abstract/Free Full Text]
    30. 30
  30. Telenti, A., N. Honore, C. Bernasconi, J. March, A. Ortega, B. Heym, H. E. Takiff, and S. T. Cole. 1997. Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J. Clin. Microbiol. 35:719-723.[Abstract]
    31. 31
  31. Telenti, A., P. Imboden, F. Marchesi, D. Lowrie, S. Cole, M. J. Colston, L. Matter, K. Schopfer, and T. Bodmer. 1993. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341:647-650.[CrossRef][Medline]
    32. 32
  32. Torres, M. J., A. Criado, J. C. Palomares, and J. Aznar. 2000. Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis. J. Clin. Microbiol. 38:3194-3199.[Abstract/Free Full Text]
    33. 33
  33. Torres, M. J., A. Criado, M. Ruiz, A. C. Llanos, J. C. Palomares, and J. Aznar. 2003. Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates. Diagn. Microbiol. Infect. Dis. 45:207-212.[CrossRef][Medline]
    34. 34
  34. van Doorn, H. R., E. C. Claas, K. E. Templeton, A. G. van der Zanden, A. te Koppele Vije, M. D. de Jong, J. Dankert, and E. J. Kuijper. 2003. Detection of a point mutation associated with high-level isoniazid resistance in Mycobacterium tuberculosis by using real-time PCR technology with 3'-minor groove binder-DNA probes. J. Clin. Microbiol. 41:4630-4635.[Abstract/Free Full Text]
    35. 35
  35. World Health Organization. 1977. Guidelines for surveillance of drug resistance in tuberculosis. Publication no. W.H.O./TB/96.216 World Health Organization, Geneva, Switzerland.
    36. 36
  36. World Health Organization. 2003. W.H.O. report 2003 global tuberculosis control. http://www.who.int/gtb/publications/globrep/index.html.

Journal of Clinical Microbiology, November 2004, p. 5277-5285, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5277-5285.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Haldar, S., Sharma, N., Gupta, V. K., Tyagi, J. S. (2009). Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR. J Med Microbiol 58: 616-624 [Abstract] [Full Text]  
  • Evans, J. T., Parveen, A., Smith, G. E., Xu, L., Chan, E. W. C., Chan, R. C. Y., Hawkey, P. M. (2009). Application of denaturing HPLC to rapidly identify rifampicin-resistant Mycobacterium tuberculosis in low- and high-prevalence areas. J Antimicrob Chemother 63: 295-301 [Abstract] [Full Text]  
  • Takahashi, T., Tamura, M., Asami, Y., Kitamura, E., Saito, K., Suzuki, T., Takahashi, S. N., Matsumoto, K., Sawada, S., Yokoyama, E., Takasu, T. (2008). Novel Wide-Range Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA: Development and Methodology. J. Clin. Microbiol. 46: 1708-1715 [Abstract] [Full Text]  
  • Takahashi, T., Tamura, M., Asami, Y., Kitamura, E., Saito, K., Suzuki, T., Takahashi, S. N., Matsumoto, K., Sawada, S., Yokoyama, E., Takasu, T. (2008). Novel Wide-Range Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA: Clinical Application for Diagnosis of Tuberculous Meningitis. J. Clin. Microbiol. 46: 1698-1707 [Abstract] [Full Text]  
  • Scott, J. C., Koylass, M. S., Stubberfield, M. R., Whatmore, A. M. (2007). Multiplex Assay Based on Single-Nucleotide Polymorphisms for Rapid Identification of Brucella Isolates at the Species Level. Appl. Environ. Microbiol. 73: 7331-7337 [Abstract] [Full Text]  
  • Bang, D., Bengard Andersen, A., Thomsen, V. O. (2006). Rapid Genotypic Detection of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Directly in Clinical Specimens.. J. Clin. Microbiol. 44: 2605-2608 [Abstract] [Full Text]  
  • Takahashi, T., Nakayama, T. (2006). Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA.. J. Clin. Microbiol. 44: 1029-1039 [Abstract] [Full Text]  
  • De Francesco, V., Margiotta, M., Zullo, A., Hassan, C., Troiani, L., Burattini, O., Stella, F., Di Leo, A., Russo, F., Marangi, S., Monno, R., Stoppino, V., Morini, S., Panella, C., Ierardi, E. (2006). Clarithromycin-Resistant Genotypes and Eradication of Helicobacter pylori. ANN INTERN MED 144: 94-100 [Abstract] [Full Text]  
  • Espy, M. J., Uhl, J. R., Sloan, L. M., Buckwalter, S. P., Jones, M. F., Vetter, E. A., Yao, J. D. C., Wengenack, N. L., Rosenblatt, J. E., Cockerill, F. R. III, Smith, T. F. (2006). Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin. Microbiol. Rev. 19: 165-256 [Abstract] [Full Text]  
  • Hazbon, M. H., Bobadilla del Valle, M., Guerrero, M. I., Varma-Basil, M., Filliol, I., Cavatore, M., Colangeli, R., Safi, H., Billman-Jacobe, H., Lavender, C., Fyfe, J., Garcia-Garcia, L., Davidow, A., Brimacombe, M., Leon, C. I., Porras, T., Bose, M., Chaves, F., Eisenach, K. D., Sifuentes-Osornio, J., Ponce de Leon, A., Cave, M. D., Alland, D. (2005). Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS6110 Clustering Rather than Ethambutol Resistance. Antimicrob. Agents Chemother. 49: 3794-3802 [Abstract] [Full Text]  
  • Espasa, M., Gonzalez-Martin, J., Alcaide, F., Aragon, L. M., Lonca, J., Manterola, J. M., Salvado, M., Tudo, G., Orus, P., Coll, P. (2005). Direct detection in clinical samples of multiple gene mutations causing resistance of Mycobacterium tuberculosis to isoniazid and rifampicin using fluorogenic probes. J Antimicrob Chemother 55: 860-865 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wada, T.
Right arrow Articles by Kobayashi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wada, T.
Right arrow Articles by Kobayashi, K.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»