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Journal of Clinical Microbiology, September 2001, p. 3350-3352, Vol. 39, No. 9
Molecular Biology Unit, Virus Reference
Division, Central Public Health Laboratory, Colindale, London, NW9
5HT,1 and PHLS Mycobacterium Reference
Unit, Dulwich Public Health Laboratory, Dulwich Hospital, London,
SE22 8QF,2 United Kingdom
Received 16 April 2001/Returned for modification 4 May
2001/Accepted 27 June 2001
A biprobe assay utilizing LightCycler technology was developed to
detect rifampin resistance-associated gene mutations in the
Mycobacterium tuberculosis rpoB gene. Three biprobes
detected all mutations present in the 46 rifampin-resistant isolates.
Wild-type sequences were correctly identified in each case. The method
was reproducible, accurate, and easy to use.
Mycobacterium
tuberculosis is responsible for three million deaths a year
worldwide (10). Recommended treatment for tuberculosis (TB) comprises a combination of four drugs: rifampin, isoniazid, pyrazinamide, and ethambutol, with or without streptomycin
(5). Resistance has emerged to all of these drugs, and
multidrug resistance (MDR), in which the isolate is resistant to
rifampin and to at least one other drug, is becoming more common
(2). As most of the MDR strains are resistant to rifampin,
the rapid detection of mutations within the rpoB gene may be
used for presumptive identification of MDR-TB (14),
allowing the correct treatment to be given promptly. Real-time
PCR and probe analysis are ideally suited to the analysis of these
mutations, allowing a rapid and accurate identification of drug
resistance to be made.
Our approach utilizes biprobes coupled with real-time PCR on the
LightCycler. The LightCycler is a real-time PCR machine that allows both rapid PCR cycling and continuous monitoring of
product formation (15). Sybr Green I, an intercalating dye
that fluoresces strongly when bound to double-stranded DNA, is included
in the reactions so that when PCR products are formed, fluorescence
increases (11). After PCR amplification the LightCycler
can monitor the melting of the DNA as temperature increases by
measuring a decrease in fluorescence as the Sybr Green I is
released. The negative derivative of fluorescence is plotted
against temperature to give a discrete melting peak.
Sequence-specific detection of the amplicons can be
achieved with a fluorescently labeled biprobe. Biprobes are
sequence-specific probes labeled with the fluorophore Cy5 and blocked
with biotin at the 3' end (1). When these probes are
included in the reaction after the melt cycle, a peak corresponding to
a decrease in fluorescence due to the release of the probe and a peak
due to melting of the PCR product can be observed. When the probe binds
to the complementary sequence in the PCR product, the Cy5 label is
excited by the energy transfer from Sybr Green I, resulting in an
increase of light emitted by Cy5. This fluorescence is measured at a
wavelength different from that emitted by Sybr Green I and so can be
distinguished. Biprobes, as well as hybridizing to perfectly
matched sequences, will also bind when a mismatch (mutation) is
present. When the biprobe binds to a mismatched sequence, a melting
temperature lower than that of a perfectly matched sequence can
be distinguished. We have designed three biprobes from the
81-bp region of the rpoB gene to detect mutations in
four codons.
We examined 47 rifampin-resistant isolates and a rifampin-sensitive
isolate of M. tuberculosis. These had been submitted to the
Public Health Laboratory Service Mycobacterium Reference Unit and identified by standard methods. DNA was extracted from cells harvested from Lowenstein-Jensen slopes as described previously (9). A 270-bp fragment was amplified with primers
rpob4 (5'-CCGCAGACGTTGATCAACA-3') and
rpob5 (5'-TACGGCGTTTCGATGAACC-3') from all
of the isolates and was sequenced using these primers according to the
manufacturer's instructions. Sequence analysis identified nine
different mutations affecting four codons within the rpoB
gene (Table 1). Of the 48 isolates
sequenced, 27 had a mutation affecting codon 531, 11 had a mutation
affecting codon 526, 7 had a mutation affecting codon 516, and 1 had a
mutation affecting codon 511. When compared to the published sequence,
the rifampin-sensitive isolate and one of the
rifampin-resistant isolates showed no mutations.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3350-3352.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of rpoB Mutations in
Mycobacterium tuberculosis by Biprobe
Analysis
ABSTRACT
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TABLE 1.
Position of mutations in isolates as determined by
DNA sequencing
A 183-bp amplicon was amplified with primers rpob1 (5'-AGGAGTTCTTCGGCACCAG-3') and rpob2 (5'-GGGTTTCGATCGGGCACAT-3') in a reaction mixture containing 1× PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl, and 2 mM magnesium chloride), a 200 µM concentration of each deoxynucleoside triphosphate, a 200 nM concentration of each primer, and 1 U of Taq DNA polymerase. After an initial denaturation step of 5 min at 95°C, PCR cycling was performed for 30 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The three biprobes were tested in a second-round, asymmetric PCR on the LightCycler using strains representing the different mutations and a rifampin-sensitive isolate. A second-round, asymmetric PCR was performed to preferentially amplify the DNA strand to which the biprobe would bind. PCRs (10 µl) contained 1 µl of the symmetric PCR product, Tris-HCl (pH 8.3) (500 mM), MgCl2 (5 mM), bovine serum albumin (1 mg/ml), deoxynucleoside triphosphates (a 200 µM concentration of each) (Gibco BRL), rpoB reverse primer (5'-G GCACGCTCGCGTGACA-3') (5 pmol/µl), Platinum Taq polymerase (0.4 U) (Gibco BRL), Sybr Green I (Bio/Gene Ltd.) diluted 0.01%, and a biprobe (5 pmol/µl). The cycling parameters used were denaturation at 95°C for 20 s and 40 amplification cycles (temperature transition rate of 20°C/s) at 95°C for 2 s, 55°C for 1 s, 60°C for 15 s, and 74°C for 10 s. PCR cycling was followed by melting-curve analysis at 40 to 99°C (temperature transition rate of 0.2°C/s) with continuous fluorescence readings.
Biprobe A (5' Cy5-CACCCAGCTGAGCCAATTC-biotin 3') spanned the
mutations at codon 511. The melt temperature of the normal sequence was
determined to be 69°C, and when a mutation was present the melt
temperature was found to be 65°C (Fig.
1A). Biprobe B (5' Cy5-TTCATGGACCAGAACAACCC-biotin 3') spanned codon 516, and when a
mutation was present at codon 516, the melt temperature was found to be
57°C, compared to 64°C in a wild-type sequence (Fig. 1B). Biprobe C
(5' Cy5-GTTGACCCACAAGCGCC-biotin 3') spanned the mutations at position 526, and three peaks were observed with this
probe. The wild-type sequence melted at 61°C, the sequence containing
a mutation at position 526 melted at 49°C, and a third melting peak
which corresponded to a mutation at codon 531 was observed at 66°C
(Fig. 1C). This third melting peak was confirmed by testing more
strains known to have this mutation and was reliably reproduced.
|
dF/dT is the first negative derivative of the change
in fluorescence (dF) divided by the change in temperature
(dT).
The presence and codon position of mutations in 48 M. tuberculosis strains determined by melting-peak analysis using the three biprobes correlated with the DNA sequence analysis. Each of the 46 resistant isolates showed an altered melting peak with only one of the three biprobes. Wild-type melting peaks were obtained with the other two biprobes for each of the 46 resistant isolates. For the rifampin-sensitive isolate and one rifampin-resistant isolate, wild-type melting peaks were observed with all three biprobes.
We have described an accurate and reproducible test for identification of the main rpoB mutations that cause rifampin resistance in M. tuberculosis. The real-time PCR assays can be completed in just 30 min, and all three probes can be run under the same cycling conditions. MDR-TB isolates are being isolated more frequently, and it is increasingly important to identify rifampin-resistant isolates rapidly. A previous study by Torres et al. (13) utilized real-time PCR and hybridization probes designed to bind to the mismatched sequence to detect two of the four mutations in the rpoB gene. Biprobes are very flexible in that they are designed to bind to perfectly matched sequences but will also bind when a mismatch is present. This allows unknown mutations to be detected. Sequencing data from other studies (3, 4, 6, 7, 8, 12) and our own data indicate that approximately 98% of mutations that give the resistant phenotype would be detected by our biprobe system. This system could also be readily applied to examination of clinical material, for example, by analyzing samples from rapid liquid cultures. LightCycler technology is also amenable to automation, making it useful for routine clinical use.
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ACKNOWLEDGMENTS |
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This work was partially funded by the Biotechnology and Biology Research Scientific Council (BBSRC).
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FOOTNOTES |
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* Corresponding author. Mailing address: Molecular Biology Unit, Virus Reference Division, Central Public Health Laboratory, 61 Colindale Ave., Colindale, London, NW9 5HT, United Kingdom. Phone: 020 8200 4400, ext. 3070. Fax: 020 8200 1569. E-mail: nsaunders{at}phls.org.uk.
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Journal of Clinical Microbiology, September 2001, p. 3350-3352, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3350-3352.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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