Abstract
We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis in an accurate, affordable, and rapid manner. Two hundred seventeen M. tuberculosis clinical isolates of known resistance phenotype were used. Twenty-nine known rpoB mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed: rpoB-F/R (for codons 516 to 539 of rpoB), rpoB-516F/R (for codons 508 to 536 of rpoB), katG-F/R (for the codon 315 region of katG), and inhA-F/R (for the nucleotide substitution of C to T at position −15 of inhA). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of rpoB. Twenty-seven of 29 known rpoB mutants were detected. In HRM curve analysis of katG and inhA, 90 INH-R strains that harbored katG or inhA mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring katG or inhA mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in M. tuberculosis in a rapid, accurate, simple, and cost-effective manner.
The rates of mortality and morbidity from tuberculosis (TB) remain high, despite intense worldwide efforts. One of the major factors sustaining the current TB epidemic is the increasing drug resistance of Mycobacterium tuberculosis strains (2). In the early 1990s, multidrug-resistant (MDR) TB cases that were resistant to at least rifampin (RIF) plus isoniazid (INH) arose (6). When the frequency and distribution of extensively drug-resistant (XDR) TB cases were assessed in 2004 by the U.S. Centers for Disease Control and Prevention and the World Health Organization, several cases of drug-resistant tuberculosis consistent with an XDR phenotype were found (7). This study revealed that 20% of the isolates met the MDR criteria; 2% of those were classifiable as XDR; and 4%, 15%, and 19% of the XDR TB cases were from the United States, South Korea, and Latvia, respectively (7). Thus, it is crucial that rapid drug susceptibility tests be developed to prevent the spread of MDR and XDR TB.
Although drug susceptibility testing (DST) is a prerequisite for accurate results, such testing requires much time and labor (3). Therefore, several molecular techniques have been applied to detect mutations related to drug resistance (5, 10). Resistance to RIF and INH, the mainstays of antituberculosis treatment, is mainly attributable to mutations in genes encoding the drug target or drug-converting enzymes (8). Early studies demonstrated that 95% of the resistance to RIF is associated with mutation of the RIF resistance-determining region of rpoB, whereas mutations in katG and the regulatory zone of inhA are most frequently associated with INH resistance (11).
The oligonucleotide chip method and real-time PCR have been used for detection of drug-resistant M. tuberculosis (17, 21, 27, 29). A novel method of high-resolution melting (HRM) curve analysis is an accurate and simple technique for analyzing the genotype without the need for specific probes. The dye LC Green, SYTO9, or Eva Green saturates amplified DNA, unlike SYBR green dye, during homogeneous melting curve analysis. Also, HRM curve analysis generates a difference plot curve, which analyzes nucleic acid sequences with high accuracy. Application of genotyping by HRM curve analysis has followed (13, 19). The aim of the study described here was to develop a useful molecular tool for the identification of drug resistance in M. tuberculosis in an accurate, rapid, and cost-effective manner.
MATERIALS AND METHODS
Strains and drug susceptibility tests.
Three groups of mycobacterial strains were collected. The first group consisted of 153 M. tuberculosis clinical isolates cultured during 2007 and 2009 in South Korea (Table 1). These have known antimicrobial susceptibility phenotypes, with 31 being RIF resistant (RIF-R), 43 being INH-R, and 102 being susceptible (S) to both drugs. The genotypes of these isolates had not been tested. The second group consisted of 44 isolates resistant to RIF or INH, or both (42 RIF-R and 37 INH-R). The genotypes of these strains were determined in a previous study (21); therefore, a bias for not choosing strains without mutations, especially for INH, was not excluded. The third group consisted of 20 INH-R strains randomly selected in order to estimate the prevalence of specific mutant types. To evaluate the ability to detect resistant strains, this study included RIF-R and INH-R strains more frequently than is usually seen clinically. The M. tuberculosis H37Rv ATCC 27294 strain (susceptible to both RIF and INH) was included as a control. The antibiotic susceptibility testing had been performed earlier at the South Korean Institute of Tuberculosis by the proportion method using Löwenstein-Jensen (L-J) medium.
TABLE 1.
Clinical isolates or DNAs of clinical isolates of M. tuberculosis used in the current study, including M. tuberculosis H37Rv
| Specimen groupa | No. of isolates |
|||
|---|---|---|---|---|
| RIF-S | RIF-R | INH-S | INH-R | |
| Group 1 | 122 | 31 | 110 | 43 |
| Group 2 | 2 | 42 | 7 | 37 |
| Group 3 | NTb | NT | 0 | 20 |
| Group 4 | 0 | 29 | NT | NT |
| Total | 124 | 102 | 117 | 100 |
Group 1, selected from clinical isolates of which drug resistance phenotypes were already known; group 2, collected from a stock of RIF- or INH-resistant strains; group 3, selected from clinical isolates resistant to INH; group 4, selected from DNAs of M. tuberculosis clinical isolates showing rare mutations in rpoB.
NT, not tested.
Extraction of DNA from the strains.
All strains were subcultured onto a slant of L-J medium. The DNA was extracted from the isolates according to a method described previously (26). In brief, one 10-μl loop of the organisms was suspended in 400 μl of TE buffer (10 mM Tris hydrochloride, 1 mM EDTA, pH 8.0). After sterilization for 20 min at 80°C, lysozyme was added to a final concentration of 1 mg/ml and the mixture was incubated for 24 h at 37°C. Next, 6 μl of proteinase K (10 mg/ml) and 70 μl of 10% sodium dodecyl sulfate were added, and incubation was continued for 10 min at 65°C. An 80-μl volume of N-acetyl-N,N,N-trimethylammonium bromide was added. The cups were vortexed briefly and incubated for 10 min at 65°C. An equal volume of chloroform-isoamyl alcohol (24:1, vol/vol) was added, and the mixture was vortexed for 10 s. After centrifugation for 5 min, a 0.6 volume of isopropanol was added to the supernatant to precipitate the DNA. After the pellet was allowed to stand for 20 min at −20°C and centrifugation for 15 min, the pellet was washed once with 70% ethanol and the air-dried pellet was dissolved in 20 μl of 0.1× Tris-EDTA buffer.
Primer design and PCR conditions.
For detecting mutations associated with RIF resistance, a 109-bp fragment containing codons 516 to 539 of rpoB was amplified using rpoB-F/R primers. Additionally, a 121-bp fragment containing mutations of codons 508 to 536 was amplified using rpoB-516F/R primers because use of the 109-bp fragment alone was not enough to detect RIF resistance in a preliminary study (data not shown). In order to detect mutations associated with INH resistance, a 120-bp fragment containing the codon 315 region of katG and a 126-bp fragment containing a nucleotide substitution of C to T at position −15 of inhA were amplified using katG-F/R and inhA-F/R primers, respectively. The primer sequences and their characteristics are shown in Table 2. Each of the PCR primers was designed using the Primer3 program (version 0.4.0; http://frodo.wi.mit.edu/primer3/). Genome DNA (5 μg) was added to 25 μl of a reaction mixture containing 5 μM each primers rpoB-F/R, rpoB516-F/R, katG-F/R, and inhA-F/R and ingredients of the Sensimix HRM kit with Eva Green dye (Quantace, London, United Kingdom). The PCR was performed on a Rotor-Gene 6000 apparatus (Corbett Life Science, Sydney, Australia). The negative control for each reaction consisted of two tubes of DNA of M. tuberculosis H37Rv. The PCR cycling was run according to the following conditions: for amplification of the 109-bp fragment of rpoB, 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 40 s; for amplification of the 121-bp fragment of rpoB, 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 40 s; and for amplification of katG and inhA, 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 40 s. All samples were tested in duplicate.
TABLE 2.
Primer sequences used to amplify rifampin and isoniazid resistance genes in M. tuberculosis
| Primer namea | Sequence | Position | Length (no. of nucleotides) | % GC content | Tmb (°C) |
|---|---|---|---|---|---|
| rpoB gene | |||||
| rpoB-F | 5′-AGC CAG CTG AGC CAA TTC AT | 2364 | 20 | 50.0 | 62.2 |
| rpoB-R | 5′-GCC CGG CAC GCT CAC GT | 2472R | 17 | 63.2 | 61.6 |
| rpoB516-F | 5′-AGG AGT TCT TCG GCA CCA G | 2347 | 19 | 63.2 | 60.4 |
| rpoB516-R | 5′-GCA CGC TCA CGT GAC AGA C | 2467R | 19 | 70.6 | 61.8 |
| katG gene | |||||
| katG-F | 5′-GGG CTG GAA GAG CTC GTA T | 2872 | 19 | 57.9 | 53.1 |
| katG-R | 5′-CCG TAC AGG ATC TCG AGG AA | 2991R | 20 | 55.0 | 53.8 |
| inhA promoter region | |||||
| inhA-F | 5′-CGT TAC GCT CGT GGA CAT AC | 164 | 20 | 55.0 | 52.9 |
| inhA-R | 5′-TCC GGT AAC CAG GAC TGA AC | 292R | 20 | 55.0 | 53.6 |
High-resolution melting curve analysis.
The HRM curve analysis was performed on a Rotor-Gene 6000 apparatus. When PCR amplification was complete, melting curves were obtained by use of an initial holding step at 60°C for 30 s, followed by a slow temperature increase at a rate of 0.3°C per second to 96°C with continuous fluorescence detection. Melting curve data were analyzed and normalized and the temperature shifts were analyzed with the Rotor-Gene 6000 series software (Corbett Life Science). The software analyzes the difference in the shape of the melting curve for a sample from the shape of the melting curve for the control strain (M. tuberculosis H37Rv) to detect sequence variants and generates a difference plot curve, which helps cluster samples into groups that have similar melting curves so sequence polymorphisms can be detected. The operator was blinded to the phenotype and genotype resistance data. All PCRs were performed in duplicate.
Sequence analysis.
The PCR products of all phenotypically resistant strains and randomly selected 64 RIF-S and 70 INH-S isolates were subjected to DNA sequence analysis, and the results were compared with those obtained with the melting curve data. After PCR, purified DNA was used as the template for sequencing PCR in a BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA). The samples were heated at 96°C for 1 min and then run for 35 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The extension products were purified using the ethanol precipitation method described in the manufacturer's manual. The pellet was rehydrated in 15 μl of formamide, heat denatured at 95°C for 5 min in a thermal cycler, and immediately put on ice for 5 min. The samples sequenced with the BigDye Terminator kit were electrophoresed on an ABI Prism 3130 instrument (Applied Biosystems).
Real-time PCR and HRM curve analysis of DNAs from known rare rpoB mutant strains.
To evaluate the ability to detect rare rpoB mutations, this study collected RIF-R isolates, mutations of which were already analyzed by PCR and sequencing (specimen group 4; 29 isolates). The DNAs had been isolated for another study and were provided by C. M. Kim. The 29 mutations are listed in Table 5.
RESULTS
Detection of rifampin resistance.
The results for specimen groups 1 and 2 are described together. The HRM curves of the 109-bp fragment of rpoB of all isolates tested were categorized into three types (Fig. 1). One type of curve was obtained from 124 RIF-S isolates, including the M. tuberculosis H37Rv strain as well as 5 phenotypically RIF-R isolates. Further sequencing analysis of these five RIF-R isolates revealed that one had a D516V mutation and four had H526D mutations. Another pattern, demonstrated in 62 RIF-R isolates, consisted of a unique melting curve differing from that of the 124 RIF-S isolates. This curve was described as the mutant type. Forty-two of 62 RIF-R isolates had S531L mutations, 11 had S531E mutations, 6 had H526Y mutations, 2 had H526L mutations, and 1 had an H526Q mutation. The remaining class of HRM curves was found in six isolates. These isolates could not be differentiated initially from the melting curves of the RIF-S isolates but could be detected by generating a suitable difference plot for the differentiation of the melting curve profiles. This curve was designated a variant type. The six isolates had D516Y mutations (Table 3).
FIG. 1.
HRM curve (a) and difference plot (b) of the 109-bp fragment of rpoB. Each line indicates the melting curve for an individual sample. In the difference plot, the curve profile of M. tuberculosis H37Rv was compared with the curve profiles of all other sample. The baseline represents M. tuberculosis H37Rv and all RIF-S isolates, as well as five RIF-R isolates. Some curves for specific mutations are indicated. The melting curves that are clearly different from those of the wild and mutant types were defined as variant-type curves. These curves are not easily differentiated from the other curves but are clearly distinguishable by a difference plot.
TABLE 3.
Results of high-resolution melting curve analyses for rpoB gene in M. tuberculosis isolates
| Phenotype | No. of strains | Mutation | Type by HRM curve analysis |
|
|---|---|---|---|---|
| 109-bp fragmenta | 121-bp fragmentb | |||
| RIF-S | 64 | Wild type | Wild | Wild |
| 60 | NTc | Wild | Wild | |
| RIF-R | 6 | D516Y (GAC → TAC) | Variantd | Mutant |
| 1e | D516V (GAC → GTC) | Wild | Wild | |
| 6 | H526Y (CAC → TAC) | Mutant | Mutant | |
| 4 | H526D (CAC → GAC) | Wild | Mutant | |
| 2 | H526L (CAC → CTC) | Mutant | Mutant | |
| 1 | H526Q (CAC → CAA) | Mutant | Mutant | |
| 14 | S531L (TCG → TTG) | Mutant | Wild | |
| 28 | S531L (TCG → TTG) | Mutant | Mutant | |
| 11 | S531E (TCG → GAG) | Mutant | Mutant | |
109-bp fragment amplification using rpoB-F/R primers covering mutations from codons 516 through 539 of rpoB.
121-bp fragment amplification using rpoB516-F/R primers covering mutations from codons 508 through 536 of rpoB.
NT, not tested.
The melting curves of this type are clearly different from those for the wild and mutant types, so we defined them as variant-type curves.
One mutant isolate was not detected by HRM curve analysis.
The HRM curves of the 121-bp fragment of rpoB detected 58 of 73 phenotypically RIF-R strains. However, 14 isolates with S531L mutations and 1 isolate with the D516V mutation were not detected (Fig. 2 and Table 3). Combining the results of the HRM curve analyses of the two amplifications of rpoB, 72 of the 73 RIF-R isolates were correctly categorized as having mutant types of the rpoB HRM curve (sensitivity, 98.6%). All 124 RIF-S isolates were categorized as wild type (specificity, 100%) (Table 3).
FIG. 2.
HRM curve (a) and difference plot (b) of the 121-bp fragment of rpoB. Each line indicates the melting curve for an individual sample. In the difference plot, the curve profile of M. tuberculosis H37Rv was compared with the curve profiles of all other samples. Some curves indicate specific mutations.
Detection of rare mutations responsible for rifampin resistance.
When it was used to test specimen group 4 for detecting rare mutations in the rpoB gene, HRM curve analysis of the 109-bp fragment of rpoB detected 25 of 29 mutants and HRM curve analysis of the 121-bp fragment detected 23 of 29 mutants. Collectively, 27 of 29 mutants could be detected (sensitivity, 93.1%; Table 4).
TABLE 4.
Results of high-resolution melting curve analyses for rpoB gene in chromosomal DNAs of M. tuberculosis
| No. of RIF-R strains (n = 29) | Mutation | Type by HRM curve analysis |
|
|---|---|---|---|
| 109-bp fragmenta | 121-bp fragmentb | ||
| 2 | L511P (CTG → CCG) | Wild | Mutant |
| 1 | L511P (CTG → CCG) | Mutant | Mutant |
| 1 | Q513P (CAA → CCA) | Mutant | Mutant |
| 1 | Q513K (CAA → AAA) | Mutant | Mutant |
| 1 | F514I (TTC → ATC) | Wild | Wild |
| 1 | M515T (ATG → ACG) | Mutant | Mutant |
| 1 | D516L (GAC → CTC) | Wild | Wild |
| 3 | H526C (CAC → TGC) | Mutant | Mutant |
| 3 | H526R (CAC → CGC) | Mutant | Mutant |
| 3 | H526P (CAC → CCC) | Mutant | Mutant |
| 1 | H526N (CAC → AAC) | Mutant | Wild |
| 2 | H526N (CAC → AAC) | Mutant | Mutant |
| 3 | H526S (CAC → AGC) | Mutant | Mutant |
| 1 | H526S (CAC → TCC) | Mutant | Mutant |
| 1 | S531W (TCG → TGG) | Mutant | Wild |
| 1 | S531W (TCG → TGG) | Mutant | Mutant |
| 2 | L533P (CTG → CCG) | Mutant | Wild |
| 1 | L533P (CTG → CCG) | Mutant | Mutant |
Detection of isoniazid resistance.
The results for specimen groups 1, 2, and 3 are described together. The 120-bp fragment of katG and the 126-bp fragment of inhA were amplified from 117 INH-S and 100 INH-R isolates. For the HRM curve of katG, three classes of melting curves were found (Fig. 3). The first type was found in all INH-S isolates as well as 32 INH-R isolates. All these 32 isolates were confirmed by sequencing analysis to have no mutation of the amplified region of katG. The second type of curve, clearly distinguished from the melting curve of the wild type, appeared in 67 INH-R isolates and was associated with S315T mutations. The third type, a variant, was demonstrated in one INH-R isolate, that with a D310A mutation (Table 5). When the inhA promoter region was amplified, two types of melting curves were generated. All 117 INH-S and 67 INH-R isolates that had no mutation of the promoter region of inhA showed the wild-type melting curve. Thirty-three INH-R isolates showed a melting curve distinguishable from that for the wild type, which had a C-to-T mutation at position −15 (Table 5). Combining the results of the two HRM curve analyses, all 90 INH-R strains that harbored katG or inhA mutations and all INH-S strains were identified correctly. Ten phenotypically INH-R strains that did not harbor any mutations in katG or inhA were not detected by HRM curve analysis.
FIG. 3.
HRM curve (a) and difference plot (b) of katG. The HRM curve indicates three different profiles. Each line indicates the melting curve for an individual sample. In the difference plot, the curve profile of M. tuberculosis H37Rv was compared with the curve profiles of all other samples. The baseline represents M. tuberculosis H37Rv and all INH-S isolates as well as isolates without mutations of katG. The mutant isolates showed melting curve profiles different from the M. tuberculosis H37Rv melting curve profile.
TABLE 5.
Results of high-resolution melting curve analysis for katG and inhA in M. tuberculosis isolates
| Phenotype | No. of strains |
katG |
inhA |
||
|---|---|---|---|---|---|
| Sequence analysis result | Type by HRM curve analysis | Sequence analysis result | Type by HRM curve analysis | ||
| INH-S | 70 | Wild | Wild | Wild | Wild |
| 47 | NTc | Wild | NT | Wild | |
| INH-R | 10 | Wild | Wild | Wild | Wild |
| 22 | Wild | Wild | −15 C to Ta | Mutant | |
| 1 | D310A (GAC → GCC) | Variantb | −15 C to T | Mutant | |
| 57 | S315T (AGC → ACC) | Mutant | Wild | Wild | |
| 10 | S315T (AGC → ACC) | Mutant | −15 C to T | Mutant | |
Nucleotide C-to-T substitution at position −15 in the promoter region of inhA.
The melting curve of this type is clearly different from the curves for the wild and mutant types, so we defined it as a variant-type curve.
NT, not tested.
To estimate the frequency of each mutation type associated with INH resistance, the results for specimen groups 1 and 3 were analyzed. (Specimen group 2 was excluded because of its possible selection bias.) All 10 isolates discordant in the phenotypic resistance and HRM curve analyses belonged to specimen group 1 or 3. Therefore, 10 of 63 INH-R isolates with no mutation in the target region of katG or inhA were categorized as wild type for both katG and inhA. The remaining 53 resistant isolates were correctly identified (sensitivity, 84.1%), being categorized as mutant or variant types of katG or inhA, or both. All 117 INH-S isolates were categorized as the wild type from both the katG and inhA HRM curves (specificity, 100%) (Table 5).
DISCUSSION
Rapid identification of drug-resistant M. tuberculosis is essential for the clinical management of tuberculosis and to minimize the transmission of MDR and XDR strains. This study evaluated the performance of HRM curve analysis as a mechanism for the rapid, accurate, and cost-effective detection of drug resistance.
All phenotypically RIF-R isolates also had mutations in rpoB sequence analysis in the current study. In HRM curve analysis of rpoB, all RIF-S isolates generated a wild-type curve (specificity, 100%) and 72 of 73 RIF-R strains were detected as mutant or variant types (sensitivity, 98.6%). This study did not detect one isolate with a D516V mutation by the current two-step analysis, resulting in a false susceptibility reading. The cause of this failure is unclear. According to DNA sequencing, all RIF-R isolates, including the one not detected by the current HRM analysis, showed mutations between codons 516 and 531. Additionally, this study confirmed that HRM curve analysis is able to detect mutations in the region between codons 516 through 533, including rare mutations responsible for RIF-R. Mutations in other regions of the gene related to RIF-R have also been reported, such as V176F and S450L (1, 4, 12, 25). However, these mutations are not frequent, and none of the clinical isolates used in the current study showed mutations in codons other than codons 516, 526, and 536 (1, 4). Therefore, we gathered some rare mutations on purpose to prove that HRM curve analysis could be applied to a wide range of mutations of rpoB related to RIF resistance, with the sensitivity being 93%.
In previous studies, the contribution of katG and inhA mutations to INH resistance differed but likely accounted for approximately 71% to 85% of INH resistance genotypes (9, 15, 21, 22). In the current study, 84% of INH-R isolates in randomly selected specimen groups (groups 1 and 3) had katG or inhA mutations, or both, which is consistent with the results of previous studies. All of these strains were correctly identified by the current HRM curve analysis. Ten phenotypically INH-R strains not harboring any mutation in katG or inhA were not detected in the HRM curve analysis. Their INH resistance may be caused by mutation of ahpC, furA, iniC, or Rv1592c (24), but it is unclear whether these strains have mutations in those genes, as we did not trace other genes in the current study. Nevertheless, the design used for this study could detect almost all major mutations harbored by katG and inhA.
In this study, a novel D310A mutation of inhA was discovered. The G309D mutation of inhA in relation to INH resistance was reported in South Korea (16), but the D310A mutation was not. It is unclear whether this mutation was causative of resistance to INH because the isolate also harbored a C-to-T mutation at position −15 in inhA. Therefore, at present, it is unclear whether detection of this mutation sorts out a resistant strain or falsely filters an insignificant mutation.
Currently, two commercial kits for line probe assays, the Genotype MTBDR and Inno-LiPA Rif kits, are commonly used for detection of resistance mutations. These kits could detect only a few specific mutations, such as katG S315T and rpoB A516G, H526D, and S531L, even though the sensitivities reached 92% to 96% (18, 28). Furthermore, these kits are expensive. In comparison with the methods that use these kits, our HRM curve analysis has some advantages, in that it can detect any mutation in the target regions. Real-time PCR usually requires specific probes, and therefore, the effort to design appropriate probes is necessary and the running cost is much higher than that of conventional PCR. However, the current method uses only a nonspecifically binding dye and does not require specific probes for real-time PCR. These advantages, i.e., low cost, no hybridization step, and simplicity in reaction and analysis, will make this method popular in diagnostic laboratories.
This study demonstrated that resistance in M. tuberculosis can be detected by HRM curve analysis. Although many molecular diagnostic tools have been developed, HRM curve analysis is simple and highly sensitive and specific. Such analysis can detect different broad-spectrum single nucleotide polymorphisms in a target gene in one step, so this method has a lower running cost than real-time PCR with a probe. Previous studies have reported that MDR in M. tuberculosis isolates was identified by rpoB scanning using HRM curve analysis. Hoek et al. (14) first reported the utility of this method using cultured M. tuberculosis isolates. In that study, the authors demonstrated very high sensitivities and specificities (94 to 98% and 96 to 100%, respectively) for detection of RIF resistance. However, the method had two steps that initially amplified products of unknown DNA which were mixed with M. tuberculosis H37Rv, followed by analysis of HRM curves. This two-step technique may cause cross contamination and may be cumbersome. Pietzka et al. reported a sensitivity of 90% for detecting RIF resistance in a small number of M. tuberculosis isolates (23). Very recently, Ong et al. (20) reported 89% and 98% sensitivities for RIF and INH resistance detection, respectively. Their method was similar to ours in using a one-step assay. Compared with the previous studies, our study tested a relatively large number of clinical isolates or DNAs originating from clinical isolates. Sensitivity and specificity were similar to the results in those studies. The one-step analysis makes it easy to apply this method in clinical microbiology laboratories. The current study also confirmed that clinical isolates can be directly applied to detect mutations associated with RIF and INH resistance in M. tuberculosis.
One limitation of the current study is that not all kinds of mutations related to RIF and INH resistance were studied, and thus, the technique cannot yet replace conventional DST. Another limitation is that the test may detect silent or other mutations not actually associated with drug resistance.
In conclusion, HRM curve analysis can detect mutations associated with RIF and INH resistance in M. tuberculosis in a simple, rapid, accurate, and cost-effective manner.
Acknowledgments
This study was supported by a grant (A080854) from the South Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea.
Footnotes
Published ahead of print on 15 September 2010.
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