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Journal of Clinical Microbiology, October 2006, p. 3826-3829, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.00225-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evaluation of Molecular-Beacon, TaqMan, and Fluorescence Resonance Energy Transfer Probes for Detection of Antibiotic Resistance-Conferring Single Nucleotide Polymorphisms in Mixed Mycobacterium tuberculosis DNA Extracts

Hasan Yesilkaya,¹ Francesca Meacci,² Stefan Niemann,³ Doris Hillemann,³ Sabine Rüsch-Gerdes,³ LONG DRUG Study Group, Michael R. Barer,¹ Peter W. Andrew,^1* and Marco R. Oggioni²

Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, United Kingdom,¹ Laboratorio di Microbiologia Molecolare e Biotecnologia, Dipartimento di Biologia Molecolare, Universit à di Siena Policlinico Le Scotte (lotto 5, piano 1), 53100 Siena, Italy,² National Reference Center for Mycobacteria, Forschungszentrum, Borstel, Germany³

Received 1 February 2006/ Returned for modification 5 April 2006/ Accepted 5 June 2006

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The ability of fluorescence resonance energy transfer, molecular-beacon, and TaqMan probes to detect single nucleotide polymorphism (SNP) in the presence of a wild-type allele was evaluated using drug resistance-conferring SNPs in mixed Mycobacterium tuberculosis DNA. It was found that both the absolute quantity and the ratio of alleles determine the detection sensitivity of the probe systems.

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Several studies have shown that single nucleotide polymorphisms (SNPs) in Mycobacterium tuberculosis confer resistance to rifampin and isoniazid (5, 15). Ninety-five percent of SNPs giving resistance to rifampin localized in a 100-bp region of rpoB (5, 14). Mutation at codon 315 of katG has been reported in a very high percentage of isoniazid-resistant isolates (1, 7, 12). Even in drug-free environments, rifampin resistance occurs at an estimated rate of 1 in 10⁸ bacilli, while isoniazid resistance arises in approximately 1 in 10⁶ bacilli (10). Consequently, mixed populations of resistant and susceptible organisms may occur even when the apparent phenotype of the culture is susceptible (14). Under selective pressure, while the susceptible bacilli are gradually eliminated, the drug-resistant bacilli multiply and become the dominant entity in the population.

Culture-based tests for drug susceptibility in M. tuberculosis may be insensitive in detection of mixed populations, and they are time-consuming (3). Furthermore, processing of clinical specimens may have a negative impact on the viability of the few resistant organisms that may be present (18). Hence, a sensitive and swift molecular method for detecting low numbers of resistant bacilli in the backdrop of drug-sensitive ones is desirable, as an inappropriate course of therapy and delayed follow-up of susceptibility tests in heteroresistance cases could permit additional resistance to develop (13).

Our aim was to investigate whether or not resistance-conferring SNPs could be identified in a rapid and efficient fashion in the presence of sensitive alleles. Real-time detection chemistries have been reported as specific and sensitive assays of SNPs. However, while many studies tested their utility in homogenous cultures or DNA solutions (2, 4, 9, 16), data on their sensitivity and specificity in detecting a single allele difference in heterogeneous DNA samples are sparse. Hence, we evaluated the usefulness of molecular-beacon, TaqMan, and fluorescence resonance energy transfer (FRET) probes in detecting resistance-conferring SNPs in the presence of the wild-type allele and vice versa.

A pair of 6-carboxyfluorescein (FAM)-labeled molecular beacons, L531mt+ and S531wt+ (Table 1), synthesized by TIB MOLBIOL (Berlin, Germany), targeted either the rifampin resistance (TTG) or wild-type (TCG) allele at codon 531 of rpoB. The 20-µl reaction mixture contained 20 µM molecular beacon, 2 mM Mg²⁺, 20 µM concentrations each of the MARF and MARR primers (Table 1), and 10 µl 2x HotStarTaq DNA polymerase kit reagents (QIAGEN, West Sussex, United Kingdom). As a source of template, threefold dilutions of 3.4 ng/µl wild-type M. tuberculosis H37Rv chromosomal DNA and of 2.13 ng/µl of chromosomal DNA of a clinical strain, designated 53-7, that was resistant to all first-line but susceptible to all second-line antimycobacterial drugs and with an SNP at codon 531 of rpoB (TCG to TTG), mixed in various ratios, were used (Table 2). The isolate was obtained from a patient who had been treated with isoniazid, rifampin, streptomycin, ethionamide, and pyrazinamide. A Stratagene MX4000 cycler was used for the reactions, and the following were the settings for PCR: 15 min at 95°C enzyme activation followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 30 s. The experiment with each probe was repeated at least four times, and the cycle number at which the signal was above the threshold fluorescence (C_T) was determined.

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TABLE 1. Primers and probes used in this study

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TABLE 2. Performance of molecular-beacon probes for the detection of a specific allele of rpoB in heterogeneous DNA solution

The TaqMan minor groove binder (MGB) probes (Table 1) were purchased from ABI (Weiterstadt, Germany) and targeted either the isoniazid resistance allele (ACC) or the wild-type allele (AGC) of codon 315 of katG (7). The 20-µl reaction mixture contained 10 µM probes, 20 µM concentrations each of katG-F and katG-R primers (Table 1), and 1x commercial ready-to-use PCR reagents (Roche, United Kingdom). As a source of template, threefold dilutions of 3.4 ng/µl wild-type M. tuberculosis H37Rv chromosomal DNA and of 2.13 ng/µl of chromosomal DNA of clinical strain 53-7, which carries an SNP at codon 315 of katG (ACC to AGC), mixed in various ratios, were used (Table 3). By mixing the two probes in equimolar ratios, the assay became a multiplex with two fluorophores producing emission maxima at separate wavelengths (FAM at 520 nm for the mutant and JOE [dichlorodimethoxyfluorescein] at 555 nm for the wild-type allele), allowing simultaneous detection of wild-type and mutant alleles in the same reaction. Real-time PCR analyses were performed in a Corbett Research Rotor Gene 2000, and the parameters for cycling were 94°C for 10 min followed by 40 cycles of 92°C for 15 s and 60°C for 1 min.

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TABLE 3. Performance of TaqMan probes for the detection of a specific allele of katG in heterogeneous DNA solution

FRET probes (Table 1) targeted either a 19-bp-long region in katG encompassing codon 315 with the OG78 probe and OG79 anchor or targeted a 15-bp mutation hot spot in the rpoB gene with the OG86 probe and OG87 anchor. The probes were designed on the wild-type sequence; thus, the presence of the mutated allele was distinguished by the difference in the melting temperatures of amplified products. The real-time PCR was done essentially as described previously (11), using amplification products of the OG76 and OG77 primers for katG and the OG84 and OG85 primers for rpoB targets (Table 1). The 20-µl reaction mixture contained 10 pmol of primers, 2 pmol of FRET probes, 4 mM MgCl₂, 1x master hybridization probes reaction mixture (Roche Diagnostics, United Kingdom), and the dilutions of 3.4 ng/µl chromosomal wild-type DNA and 2.13 ng/µl mutated DNA, with SNPs in either the rpoB gene or the katG gene as described above. The PCR cycling parameters consisted of an initial denaturation step at 95°C for 30 s, followed by 40 cycles of repeated denaturation (0 s at 95°C), annealing (10 s at 57°C), and polymerization (10 s at 72°C). The temperature transition rate was 20°C/s in all segments. In the final cycle, the melting curve was determined in the samples by initial heating to 95°C, cooling to 45°C, and subsequent controlled heating to 95°C with a temperature transition rate of 0.1°C/s.

Molecular-beacon and TaqMan probes. The results with molecular-beacon and TaqMan probes showed that when only a single mutant or wild-type allele was present, the C_T value decreased with an increasing amount of initial template quantity, as expected. An amplification signal could be obtained with the lowest template quantity that was tested, which was approximately 3 to 5 pg for both alleles, with either probe system (Tables 2 and 3). When both alleles were present in the reaction, the detection ability was determined first by the amount of each allele and second by the ratio of alleles, irrespective of the probe system (Tables 2 and 3). Regardless of whether a second allele was present, detection of both mutant and wild-type alleles was achieved consistently with both methodologies when the reaction mixtures contained between 0.126 and 3.4 ng DNA for the wild-type allele and 0.079 and 2.13 ng DNA for the mutant allele (Tables 2 and 3). In 16 reactions with each method, DNA ratios ranged from 1 to 43, and in 14 reactions both alleles could be detected, provided the ratios of the alleles did not exceed 1:5.6 (Table 4). The only exception to this was the detection of a 1:14 ratio with the TaqMan probe (Tables 2 and 4).

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TABLE 4. Summary of detection of specific SNPs in heterogeneous DNA samples by TaqMan and molecular-beacon probes

With both systems, in mixtures containing low quantities of templates (0.0047 to 0.042 ng of the wild-type allele and 0.0029 to 0.026 ng of the mutant allele), both alleles could be detected provided the ratio of alleles did not exceed 1:4.8 (Tables 2, 3, and 4). In all other cases, only the higher-quantity allele could be detected (Tables 2, 3, and 4). This shows that the ratio of the alleles adversely affected the detection ability of both probe systems.

The presence of a second allele did not influence the specificity of probe systems. This was evident from the fact that similar C_T values were obtained for mixed and homogenous reactions containing the same quantity of target DNA. For example, while the C_T value of reactions containing 3.4 ng wild-type allele and up to 2.13 ng mutant allele ranged from 25.6 to 24.5, the C_T value of reactions containing only 3.4 ng wild-type DNA was 25.2 (Table 2). The differences in observed C_T values were not statistically significant (P > 0.05).

FRET probes. When the chromosomal DNA mixtures were tested with FRET probes, no amplification signal could be detected whatever the quantity or ratio, even though in reactions containing solely wild-type or mutant allele successful amplification was obtained with as little as 3 to 5 pg for both alleles. This could be due to interference by high-molecular-weight nonspecific chromosomal DNA. Indeed, it was only after a preamplification step that the detection of either allele alone could be achieved to a sensitivity similar to that of the molecular-beacon and TaqMan probes.

The diagnostic value of SNP analysis in the presence of a wild-type DNA is good not only for speedy detection of drug-resistant M. tuberculosis but also in nonmicrobiological situations, for example, the diagnosis of SNPs associated with biologically significant phenotypic traits, such as cancer and mitochondrial diseases (6, 17). Although several methods are available for SNP detection in mixed DNA samples, for example, bioluminometric assay coupled to modified primer extension reaction and DNA microarray technology (8), the requirement for multiple procedures and the costs limit their routine use. In this study, we demonstrated the use of fluorogenic allele-specific detection probes that allow PCR amplification and allele detection in a single procedure. It was shown that molecular-beacon and TaqMan probes can specifically detect individual alleles in a mixture, but this was governed by the ratio of the alleles as well as the absolute quantity of each allele. We found that FRET probes can also reach the detection level of molecular beacons and TaqMan probe systems but only after target enrichment by primary PCR, suggesting that it may be a less useful technique here.

The implication of our results is that these techniques can be used for detection of heteroresistance earlier than is possible with culture. However, the requirement to design individual probes for each SNP increases the costs. Consequently, beacon and TaqMan probes may be best suited for detecting commonly encountered SNPs, such as those in codon 315 of katG or codon 531 of the rpoB gene. On the other hand, although FRET probes required two rounds of PCR amplification, they are ideal for initial screening of multiple SNPs and they are more cost efficient for high-throughput screening. Preliminary data on field application of these three approaches indicate a detection limit near that of phenotypic drug susceptibility testing and hence their usefulness as molecular tools.

 
This LONG DRUG project was supported by the European Union (QLK-CT-2002-01612).

 
* Corresponding author. Mailing address: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, United Kingdom. Phone: (44) 116 2522941. Fax: (44) 116 2525030. E-mail: pwa{at}le.ac.uk.

The LONG DRUG study group is composed of Marco R. Oggioni and Francesca Meacci, C Trappetti Università di Siena; Francesco Checchi and Maryline Bonnet, Epicentre Paris; Graziella Orefici, Manuela Pardini, and Lanfranco Fattorini, Istituto Superiore di Sanità Roma; Peter Andrew, Mike Barer, and Hasan Yesilkaya, University of Leicester; Heinz Rinder, University of München; Sabine Rüsch-Gerdes and Stefan Niemann, Research Centre Borstel; Germano Orrù, Università di Cagliari; Francis Varaine, Médecins Sans Frontières Paris; and Thierry Jarosz, 3Es Paris.

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Journal of Clinical Microbiology, October 2006, p. 3826-3829, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.00225-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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