Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout
Herthnek D (2013) Detection of Rifampicin Resistance in Mycobacterium tuberculosis by
Padlock Probes and Magnetic Nanobead-Based Readout. PLoS ONE 8(4): e62015. doi:10.1371/journal.pone.0062015
Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead- Based Readout
Anna Engstro m 0
Teresa Zarda n Go mez de la Torre 0
Maria Strmme 0
Mats Nilsson 0
David Herthnek 0
Igor Mokrousov, St. Petersburg Pasteur Institute, Russian Federation
0 1 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden, 2 Department of Preparedness, Swedish Institute for Communicable Disease Control, Solna, Sweden, 3 Department of Engineering Sciences, Division of Nanotechnology and Functional Materials, Uppsala University, The A ngstro m Laboratory, Uppsala, Sweden, 4 Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden, 5 Department of Immunology , Genetics and Pathology , Uppsala University, Science for Life Laboratory, Rudbeck Laboratory , Uppsala , Sweden
Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.
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Funding: The work was funded by the Carl Trygger Foundation, FORMAS (the Swedish Research Council (VR)), VINNOVA (Swedish Governmental Agency for
Innovation Systems), and the Innovative Medicines Initiative, a public-private partnership between the European Union, and the European Federation of
Pharmacetical Industries and Associations (RAPP-ID project, grant agreement, no. 115153). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have read the journals policy and have the following conflicts: MN holds stock in the company Olink Bioscience that holds
commercial rights to padlock probes. MS and MN holds parts of a patent on the VAM-NDA technique. This does not alter the authors adherence to all the PLOS
ONE policies on sharing data and materials.
. These authors contributed equally to this work.
Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains
as a major public health problem. Increasing resistance to anti-TB
drugs severely threatens the control of the disease. Prompt
detection of drug-resistant M. tuberculosis strains is crucial for
prescription of appropriate treatment. However, conventional
drug susceptibility testing is a very time-consuming procedure,
requiring weeks to months to complete due to the slow growth of
the causative agent. In order to quickly detect drug-resistant M.
tuberculosis it is therefore essential to use molecular based diagnostic
methods, which can be performed within a day.
Chromosomal mutations are the genetic basis for drug
resistance in M. tuberculosis [1,2]. The effective first line anti-TB
drug rifampicin (RIF) inhibits transcription by binding to the
subunit (encoded by rpoB) of the RNA polymerase [3]. Resistance
to RIF in M. tuberculosis is almost entirely associated with mutations
within an 81-bp region of the rpoB gene, called the RIF
resistancedetermining region (RRDR), comprising codons 507 to 533 [2,4].
A substitution in the first or second nucleotide position of codons
516 and 526 or in the second nucleotide position in codon 531 are
the most commonly observed mutations in RIF-resistant clinical
isolates of M. tuberculosis [5].
Padlock probes are linear oligonucleotides that comprise two
target-specific sequences at the 39 and 59 ends, and a linker
segment containing sequences for amplification and detection [6].
The padlock probe ends are brought into juxtaposition upon
hybridization to the target sequence, allowing padlock probe
circularization by ligation. A mismatch near the ligation junction,
and in particular at the 39 end of the probe, prevents ligation [7],
providing a specific means of mutation detection. Padlock probes
were chosen due to this promising virtue of specificity and ease of
multiplexing [8,9,10,11], while the various strategies to improve
the specificity of allele-specific PCR are often insufficient,
elaborate to design and can lead to compromised amplification
efficiency [12,13]. Isothermal signal amplification of the padlock
probe is achieved through rolling circle amplification (RCA) of the
reacted padlock probes [14,15]. The concatemer of replicated
padlock probe produced by RCA is restriction digested, re-ligated
into new circles and subjected to an additional round of RCA,
known as circle-to-circle amplification (C2CA) [8], increasing the
sensitivity of the assay.
Nanomedicine is just starting to reshape clinical practice [16]
and since last decade magnetic nanoparticles, or so called
nanobeads, have drawn increasing attention to the development
of different types of magnetic biosensors. This is because of their
high physical and chemical stability and that they are not generally
affected by reagent chemistry or exposure to light. Magnetic
nanobead-based biosensors offer an attractive and cost-effective
route for detection of biomolecules, since they are relatively
inexpensive to produce and easily made biocompatible [17]. The
portable readout instrument for the detection principle described
below should be possible to produce inexpensively, as it contains
no optics but only electromagnetic device components. The
Brownian relaxation principle constitutes a substrate-free
biosensor method, where suspended magnetic nanobeads exhibiting
Brownian relaxation behavior [18] are equipped with probe
molecules for recognition of specific target molecules.
Hybridization of target molecules to the probes causes a hydrodynamic size
increase of the nanobeads, corresponding to the size of the target
molecule. This brings on a decrease in the relaxation frequency of
the beads, defined by the position of the peak in the imaginary part
x of the complex magnetization spectrum x~x{ix, since
the frequency is inversely proportional to the hydrodynamic size of
the beads. The concentration of the targets can be monitored as a
corresponding decrease of the amplitude of the relaxation peak
xmax of the free beads [19], and several targets can, in principle,
be monitored simultaneously by employing differently sized beads
[20]. This strategy is employed in the volume amplified magnetic
nanobead detection assay (VAM-NDA) [19,21,22,23].
We have developed a molecular method for detection of RIF
resistance in M. tuberculosis by padlocks probes, RCA and a
magnetic nanobead-based readout; VAM-NDA. The nine most
common rpoB mutations, located in codons 516, 526 and 531,
were targeted by a cocktail of padlock probes, making use of their
high suitability for multiplexing [8]. The assay includes a probe for
species detection and a novel type of padlock probe system
confirming loss of wild type at any of the three investigated RRDR
codons.
Materials and Methods
Bacteria, DNA Extraction and Fragmentation
The reference strains M. tuberculosis H37Rv (ATCC 25618), M.
avium (ATCC 25291), M. marinum (ATCC 2275), M. microti (ATCC
19422), and clinical isolates M. interjectum s99/96, M. kansasaii Alk
Prague, M. szulgai BTB 98-526, M. canetti BTB 04-106, M. bovis
BTB 08-329, M. africanum XTB 08-066 were included in the study.
In addition, eight RIF-resistant M. tuberculosis clinical isolates, each
harboring a 516 TAC, 516 GTC, 526 CGC, 526 CTC, 526 AAC,
526 TAC, 531 TTG or 531 TGG rpoB mutation, were also
included [24]. Bacteria were cultured on Lo wenstein-Jensen (LJ)
medium or on LJ-medium containing 40 mg/L RIF prior to DNA
extraction, as previously described [25]. Ten micrograms of
genomic DNA was fragmented by 10 U each of NaeI and
HpyCH4V (New England Biolabs, Ipswich, MA, USA) at 37uC
for 90 min, following enzyme inactivation at 65uC for 20 min.
DNA concentration was measured by Qubit dsDNA HS and BR
assays (Invitrogen, Carlsbad, CA, USA).
Padlock Probes and Oligonucleotides
Sequences were obtained from the M. tuberculosis H37Rv
genome (GenBank accession no. NC_000962; NCBI bank) [26]
and the Tuberculosis Drug Resistance Mutation Database [5].
Sequences of padlock probes and oligonucleotides used in the
study (Integrated DNA Technologies, Inc.,Coralville, IA, USA)
are specified in Table S1 in the supporting information. A wild
type padlock probe system consisting of a padlock probe and two
gap-fill oligonucleotides (P5875, L11420, L11170) was designed to
hybridize to rpoB codons 511 to 534 with ligation sites at the first
nucleotide positions of codons 516 and 526, and the second
nucleotide position of codon 531. Nine mutant-specific padlock
probes were designed for detection of mutations at the first and
second nucleotide positions of rpoB codons 516 and 526, and at the
second nucleotide position of rpoB codon 531. Mutant-specific
probes targeting the same nucleotide position but different
nucleotides were ordered degenerated at the 39 end nucleotide
position of the probe (526 CKC, 526 DAC and 531 TKG). A
padlock probe was designed for the 16S23S internal transcribed
spacer (ITS) region for detection of the M. tuberculosis complex
(MTC). The hybridizing padlock probe arms were designed to
have salt adjusted melting temperatures (Tm) between 50uC and
60uC, as calculated by the online software OligoCalc version 3.26
[27]. The backbone of the padlock probes linking the arms
together consisted of hybridization sites for restriction
oligonucleotides [28] and detection oligonucleotides. Due to possible
coincidental matching of backbone with arms or disposition for
folding due to high GC content [29], predictions of secondary
structures were made using the online tool Mfold Web Server [30]
with default settings. Efforts were made to avoid structures with
low free energy (DG) having high probability of forming [31], by
adjusting the lengths of the arms.
Padlock probes and the gap-fill oligonucleotides L11420,
L11170 and L11171 were phosphorylated at the 59 end by mixing
1 mM oligonucleotide with 16 PNK buffer A, 1 mM ATP
(Fermentas, Vilnius, Lithuania), and 1 U/ml T4 polynucleotide
kinase (Fermentas), and incubating at 37uC for 30 min followed by
enzyme inactivation at 65uC for 20 min. The phosphorylated
oligonucleotides were stored at 220uC until use.
Padlock Probe Ligation and Amplification
Target recognition and amplification by C2CA was performed
essentially as previously described [32]. Three nanogram of
genomic DNA were used for specificity testing of the wild type
probe system, the mutant-specific padlock probes, and the M.
tuberculosis complex padlock probe. Thirty nanogram of genomic
DNA were used for the final test of the complete assay. One
microliter of template DNA (synthetic or genomic) was added to a
19 ml ligation mixture consisting of 16 ligase buffer (see below),
250 mU/ml Ampligase (EpiCenter, Madison, WI, USA) or Tth
DNA ligase (GeneCraft, Cologne, Germany), 0.2 mg/ml Bovine
Serum Albumin (BSA, New England Biolabs), 33 nM of each
phosphorylated padlock probe, or 100 nM of phosphorylated
P4782, P4949, or P5875, and 50 nM of the capture probe. The
gap-fill oligonucleotides were added in 50 nM to the wild type
probe system reactions. When Ampligase was applied, the ligase
buffer comprised of 18 mM TRIS-HCl pH 8.3, 22.5 mM KCl
(Merck, Whitehouse Station, NJ, USA), 9 mM MgCl2
(SigmaAldrich, St. Loius, MO, USA), 0.009% Triton X-100
(SigmaAldrich) and 0.5 mM NAD (Sigma-Aldrich), and when
GeneCrafts Tth DNA ligase was applied, the included reaction buffer
was used. Genomic DNA was denaturated at 95uC for 4 min prior
to use. Hybridization and ligation were performed at 60uC for
5 min. MyOne Dynabeads T1 (Invitrogen) were washed three
times in 16 Wtw buffer (10 mM TRIS-HCl pH 7.5, 5 mM
EDTA, 0.1% Tween 20 [Sigma-Aldrich], 0.1 M NaCl).
Unreacted padlock probes were removed by capture of the target
DNA to 50 mg Dynabeads by coupling to the biotinylated capture
oligonucleotide followed by a wash with 16 Wtw buffer using a
permanent magnet. Twenty microliters of RCA mixture
containing 0.2 mg/ml BSA, 125 mM dNTP (Fermentas), 16 Phi29 buffer
(Fermentas) and 100 mU/ml Phi29 DNA polymerase (Fermentas)
was added to the Dynabeads. RCA was performed at 37uC for
20 min followed by enzyme inactivation at 65uC for 1 min. To
monomerize the RCA products, restriction oligonucleotide
L12890 (120 nM) was added with 0.2 mg/ml BSA and 120 mU/
ml of AluI (New England Biolabs) in 16 Phi29 buffer followed by
incubation at 37uC for 1 min and enzyme inactivation at 65uC for
1 min. After the beads were discarded, the monomerized RCA
products were re-circularized and amplified by addition of 16
Phi29 buffer, 0.2 mg/ml BSA, 100 mM dNTP, 0.68 mM ATP,
60 mU/ml Phi29 DNA polymerase, 14 mU/ml T4 DNA ligase
(Fermentas) and incubated at 37uC for 20 min followed by enzyme
inactivation at 65uC for 1 min.
Improvements of the Wild Type Probe System
Initial probe design was compared to improved
oligonucleotides by applying different sets of wild type probe components
on 10 amol synthetic target DNA (L10919). The padlock probe
and the longer gap-fill oligonucleotide were stepwise exchanged
to the improved versions (P4782 to P4949, and L11171 to
L11420). For concordance with the detection oligonucleotide
used by the other probes, P4949 was subsequently modified to
P5875.
Digital Quantification of RCA Products
RCA products were quantified using a single-molecule detection
method [33] (herein referred to as SMD), as briefly described
below. A fluorescent dye-coupled oligonucleotide (L13179, 5 nM,
except for the wild type probe system improvements test described
above where L10806 was used) was hybridized to the RCA
products in a reaction mixture consisting of 20 mM EDTA,
20 mM TRIS-HCl, 0.1% Tween 20 and 1 M NaCl, for 2 min at
70uC and 15 min at 55uC. The sample was pumped through a
microchannel mounted on a standard confocal fluorescence
microscope operating in a line-scan mode, and individual RCA
products were detected and quantified in Matlab 7.0 (MathWorks,
MA, USA).
Coupling of Oligonucleotides to Magnetic Nanobeads
Two hundred microliters of spherical avidin-functionalized
magnetic nanobeads (Micromod Partikeltechnologie GmbH,
Rostock, Germany) with a nominal bead diameter of 100 nm
was washed twice with 16 Wtw buffer using a permanent
magnet. The beads were thereafter resuspended in 100 ml 16
Wtw buffer and incubated with 20 ml of 10 mM
biotinconjugated oligonucleotides (L9261) for 30 min at room
temperature. The beads were washed twice and resuspended
in 200 ml 16 PBS (pH 7.5).
Magnetic Bead-based Readout
After dilution of the oligonucleotide-tagged beads to a
concentration of 2 mg/ml, 20 ml was added to 100 ml of RCA
products. The solution was incubated for 20 min at 60uC and
thereafter diluted with 80 ml of a mixture containing equal volume
of 16 PBS (pH 7.5) and a solution containing 20 mM EDTA,
20 mM TRIS-HCl, 0.1% Tween 20 and 1 M NaCl.
Measurement of the frequency-dependent magnetic response at 24uC was
performed in a DynoMagH-instrument (Acreo, Sweden, frequency
range 1 Hz100 kHz and AC field amplitude 0.5 mT). It should
be noted that there were unavoidable variations in magnetic
material in each sample. Therefore, in order to normalize the
magnetic response with respect to the amount of magnetic
material in each sample, the data was normalized against the
constant value of the in-phase component of the volume
susceptibility (x?) as this value is proportional to the total content
of iron-oxide nanoparticles in a sample as previously described
[23]. The x? was measured at frequencies well above the
Brownian relaxation frequency. In the current work, the limit of
detection (LOD) was defined as the lowest tested amount of DNA
yielding a normalized magnetic response differing more than three
standard deviations of the negative control.
A molecular method for detection of RIF-resistant M. tuberculosis
was developed. The method is outlined in Figure 1 and consists of
a novel wild type padlock probe system comprising a padlock
probe and two gap-fill oligonucleotides, a cocktail of nine
mutantspecific padlock probes for detection of mutations in rpoB codons
516, 526 and 531, and an M. tuberculosis complex (MTC)-specific
padlock probe. Efficiency, specificity and multiplexing of probes
were investigated by the SMD method, and the complete assay
was read out by the VAM-NDA using a portable AC
susceptometer.
Development of the Wild Type Probe System
In order to confirm loss of wild type due to presence of any of
the mutations detected by the mutation-specific padlocks, a novel
design for analyzing several codons simultaneously was developed.
A padlock probe was designed to hybridize upstream of codon 516
and downstream of codon 531, with its 39 end targeting specifically
the second nucleotide position of codon 531, the most commonly
mutated nucleotide in RIF-resistant M. tuberculosis [2,5]. In
between the padlock probe arms, two gap-filling oligonucleotides
were designed with their 39 ends targeting the first nucleotide of
codons 516 and 526. For the wild type probe system to be able to
circularize in spite of the long rigid duplex [34] formed by the
three target-hybridizing oligonucleotides, the originally 38
nucleotides long backbone was extended by poly-T spacers to 48
nucleotides while the padlock arms were shortened, reducing the
duplex length from 80 to 72 nucleotides. To relieve strain in the
duplex further, the longest gap-fill oligonucleotide (between
codons 516 and 526) was designed to contain one deletion and
two deliberate target mismatches to make it act as a hinge. Both
these measures increased the efficacy of the system significantly
(Figure S1). Each of the improved probe system components alone
increased the product yield by more than 10 times. Together, they
increased the signal more than 60 times.
Specificity Testing of Wild Type Probe System
Specificity of the wild type probe system was evaluated on DNA
samples from a collection of M. tuberculosis strains. These include
the RIF-susceptible reference strain M. tuberculosis H37Rv,
harboring a wild type rpoB gene, and eight RIF-resistant M.
tuberculosis clinical isolates, harboring a mutation at the first or
second nucleotide position of rpoB codons 516 and 526, or a
mutation at the second nucleotide position of rpoB codon 531. The
ratios between the signals of matching (wild type rpoB) and
mismatching (mutant rpoB) target ranged from 51 (rpoB 531 TTG
mutation) to 830 (rpoB 516 GTC mutation) (Figure 2),
demonstrating robust discrimination between the wild type and mutant
strains.
Figure 1. Outline of the molecular method. A DNA sample is distributed into three tubes containing (1) the wild type padlock probe system (a
padlock probe and two gap-fill oligonucleotides with ligation sites at rpoB codons 516, 526 and 531), (2) a cocktail of nine mutant-specific padlock
probes for detection of mutations at codons 516, 526 and 531, and (3) an M. tuberculosis complex (MTC)-specific padlock probe targeting the 16S23S
intergenic spacer region (ITS). For each probe system, there was also a (4) template negative control. After hybridization and ligation of the padlock
probes, the circularized probes are amplified by rolling circle amplification (RCA). Oligonucleotide-conjugated magnetic nanobeads are added to
each tube, and the frequency-dependent magnetic response is measured. The RCA products are detected by the decrease in the Brownian relaxation
magnetic susceptibility peak of non-coupled beads. A: a wild type rpoB DNA sample yields signal only in tube 1 and tube 3. B: a DNA sample with
mutation in rpoB codon 531 yields signal only in tube 2 and 3.
doi:10.1371/journal.pone.0062015.g001
Testing of Specificity and Multiplexability of
Mutantspecific Padlock Probes
Specificity and multiplexability of the nine mutant-specific
padlock probes were tested on their respective matching target
(mutant rpoB) and on mismatching target (wild type rpoB) (Figure 3).
All probes were assessed to be specific, with a matching/
mismatching signal ratio ranging from 61 (526 AAC probe;
Figure 3C) to 332 (516 GTC probe; Figure 3A). Efficiency of
probes was compared between single-, du-, or triplex reactions to
the full nine-plex assay. The mutant-specific padlock probes that
were designed to target the same nucleotide position but different
nucleotides were synthesized degenerated at the 39 end nucleotide
of the probe (526 CKC, 526 DAC and 531 TKG), thus these
probes could not be evaluated in a singleplex format, but were
instead evaluated in a duplex (526 CKC and 531 TKG) or triplex
(526 DAC) format. The efficiency reduction of nine-plex reactions
(Figure 3) was marginal (maximum 2-fold signal decrease) and all
probes yielded a comparable signal on their respective matching
target.
Specificity Testing of the M. tuberculosis Complex Probe
The M. tuberculosis complex padlock probe targeting the 16S
23S ITS region proved to be specific when tested on a range of
mycobacterial species, including five members of the M. tuberculosis
complex; M. africanum, M. bovis, M. canetti, M. microti and M.
tuberculosis, and five nontuberculous mycobacterial species; M.
Figure 2. Specificity testing of wild type probe system. The system was tested on the reference strain M. tuberculosis H37Rv (representing wild
type rpoB), eight M. tuberculosis clinical isolates, each harboring an rpoB 516 TAC, 516 GTC, 526 CGC, 526 CTC, 526 TAC, 526 AAC, 531 TTG or 531 TGG
mutation, and a synthetic target representing an rpoB 526 GAC mutation. RCA products were hybridized to fluorescent dye-coupled oligonucleotide
and visualized in a confocal microscope. NC: negative control.
doi:10.1371/journal.pone.0062015.g002
avium, M. interjectum, M. kansasaii, M. marinum, M. szulgai (Figure 4).
The matching/mismatching signal ratio was greater than 2500.
Magnetic Bead-based Readout
LOD of the magnetic readout was determined with the wild
type probe system on synthetic target representing a wild type
RRDR. The LOD was determined to 10 amol target molecules
(Figure S2). The complete assay was evaluated on four DNA
samples; the RIF-susceptible reference strain M. tuberculosis H37R
(wild type RRDR), and three RIF-resistant M. tuberculosis clinical
isolates harboring an rpoB 516 GTC, 526 TAC or 531 TTG
mutation. The assay successfully identified all four samples; the
presence of M. tuberculosis complex DNA with, either a wild type
RRDR, or a mutant version of the locus (Figure 5), without
yielding false-positive signal from a mismatching probe, when
compared to the signals of the negative controls. The xmax values
were obtained from the complex susceptibility curves displayed in
Figures S3S4 in the supporting information.
We have developed a molecular method for detection of RIF
resistance in M. tuberculosis by padlock probes and a magnetic
bead-based readout (Figure 1). Padlock probes were designed to
target the wild type RRDR region of rpoB, and the most frequent
mutations giving rise to RIF resistance, i.e. at the first and second
nucleotide positions of codons 516 and 526, and at the second
nucleotide position of codon 531. A padlock probe for detection of
the M. tuberculosis complex targeting the 16S23S ITS region was
also included in the assay. Ligated padlock probes were amplified
by RCA and the VAM-NDA was used as a readout format.
The efficiency of the wild type probe system was improved by
shortening the padlock probe arms, prolonging the backbone, and
introducing a deletion and two mismatches in the longest gap-fill
oligonucleotide. The great effect of this relatively slight change of
the proportion of single-stranded and double-stranded parts of the
DNA circle suggests that the original probe system was on the
verge of not being able to bend and circularize at all. As the wild
type probe system is still not as efficient as the single padlock
probes, additional spacer nucleotides could probably increase the
efficiency further. This example of an oligonucleotide assembly
demonstrates the importance of accounting for tension in the
duplex structure of circularizing or folding DNA probes. The new
type of padlock probe system that we describe here could be used
to investigate DNA sequences of other species and in other
contexts where multiple variable bases are closely located in the
target sequence.
The wild type probe system was shown to be specific for wild
type target and to discriminate against all nine rpoB single
nucleotide variants investigated (Figure 2). This strongly indicates
that not only a single nucleotide mismatch at the very 39 end of the
padlock probe, but also that a mismatch at the position adjacent to
the 39 end prevents ligation. This was demonstrated by the lack of
Figure 3. Specificity and multiplexability testing of the mutant-specific padlock probes. The probes were tested in single-, du-, or tri-plex
and nine-plex (all mutant-specific padlock probes) on eight M. tuberculosis clinical isolates harboring an rpoB 516 TAC or GTC mutation (A), an rpoB
526 CGC or 526 CTC mutation (B), an rpoB 526 AAC or 526 TAC mutation (C), an rpoB 531 TTG or 531 TGG mutation (D), on a synthetic target with an
rpoB 526 GAC mutation (C), and on the reference strain M. tuberculosis H37Rv (representing wild type rpoB) (AD). RCA products were hybridized to
fluorescent dye-coupled oligonucleotide and visualized in a confocal microscope. NC: negative control.
doi:10.1371/journal.pone.0062015.g003
signal for rpoB targets harboring a 516 GTC, 526 CGC or 526
CTC mutation. In conclusion, the wild type padlock probe system
will only generate a signal if all positions are wild type and hence,
lack of signal demonstrates loss of wild type at any of the three
codons. In the very rare occasion where a mutation is present at
the first or third nucleotide position of codon 531, and at the third
nucleotide position of codons 516 and 526 [5], it is likely that
ligation would be greatly hindered and thus not yield a signal
either.
Initially, all nine mutant-specific padlock probes were designed
to target the same DNA strand, and despite efficient hybridization
and ligation, it could be shown that the signal from the 516 GTC
probe was greatly reduced when tested in the multiplex assay
(Figure S5). We hypothesized that this reduction was due to
blockage of target-primed RCA initiation by hybridization of
downstream padlock probes targeting codons 526 and 531.
Padlock probes designed for codons 516 and 531 do not compete
for the same target sequencing binding site, but may block the 59
39 exonucleolytic digestion by phi29 polymerase that is required to
acquire a free 39 end adjacent to the ligated padlock for
targetpriming of the RCA process. In order to circumvent this problem
during the multiplexed assay, the mutant-specific padlock probes
Figure 4. Specificity testing of the M. tuberculosis complex padlock probe. The probe, designed to target the 16S23S intergenic spacer
region, was tested on five M. tuberculosis complex species; M. africanum, M. bovis, M. canetti, M. microti and M. tuberculosis, and on five
nontuberculous mycobacterial species; M. avium, M. interjectum, M. kansasaii, M. marinum, M. szulgai. RCA products were hybridized to fluorescent
dye-coupled oligonucleotide and visualized in a confocal microscope. NC: negative control.
doi:10.1371/journal.pone.0062015.g004
targeting codon 516 were designed to target the coding DNA
strand while the probes targeting codon 526 and 531 were
designed to target the non-coding DNA strand.
The specificity and multiplexability of the nine mutant-specific
padlock probes were evaluated on DNA extracted from eight
RIFresistant M. tuberculosis clinical isolates (harboring a mutation in
either codon 516, 526 or 531), and on a synthetic target
(representing the rpoB 526 GAC mutation) (Figure 3). All probes
were shown to be specific for discrimination between their
respective single nucleotide variant and the wild type sequence.
In addition, the number of RCA products was not considerably
affected when the reactions were performed nine-plex compared
to single-, du-, or triplex (Figure 3). Notably, padlock probes
designed for codon 526 and 531 seem to compete with each other
for hybridization rather than blocking each other. This shows that
the assay is highly flexible and multiplexable. For further extension
of the method, more mutation-specific padlock probes could be
added to the system. Ideally, a molecular method designed to
detect mutations associated with drug resistance in M. tuberculosis
should be both flexible and multiplexable in order to act in
accordance with the continuously gained understanding of M.
tuberculosis drug resistance and to address the different needs of
assay design in various global settings.
During the probe design phase it was occasionally observed that
hybridization, and subsequent ligation, of some probe versions
were less efficient than others (as determined by the SMD
method). The M. tuberculosis genome contains 65% GC [29], and
the RRDR itself is known to form complicated secondary
structures in form of hairpins [35]. It can be hypothesized that
some of the original probes were not efficient due to secondary
structure formation, probably both in the probe itself and in the
single stranded DNA target. PCR-based methods have the
advantage that the primers can be moved to a more AT-rich
region for more efficient hybridization; however, due to the
specific approach of single nucleotide variant detection used in this
study, the probes could not be moved. Probe hybridization and
ligation was improved for suboptimally performing probes by
varying probe arm length, until satisfactory performance was
achieved (Figure S6).
Evaluation of LOD for the magnetic readout format was
performed with the wild type probe system since it yielded the
lowest signal as determined by the SMD method (Figure 2)
compared to the mutant-specific padlock probes (Figure 3). This is
conceivable, since the wild type probe system requires
hybridization of several oligonucleotides and three ligation events. The
complete assay with the VAM-NDA as readout format was
evaluated on four M. tuberculosis DNA samples. One was obtained
from a RIF-susceptible strain and contains a wild type RRDR,
while three were obtained from RIF-resistant strains, each
harboring the most prevalent mutation in each of the investigated
codons, i.e.: 516 GTC, 526 TAC and 531 TTG. All four samples
were correctly identified by the assay as M. tuberculosis complex
DNA, with either a wild type or a mutant version of RRDR
(Figure 5). The wild type probe system yielded only signal in
presence of wild type RRDR, and in the reversed scenario, the
mutant-specific padlock probe cocktail yielded only signal in
presence of a mutant version of RRDR. The assay was not only
able to identify the most prevalent mutations in rpoB responsible
for RIF-resistance in M. tuberculosis, but also confirmed loss of wild
type and detected M. tuberculosis complex DNA. A mutation in the
RRDR elsewhere than at the nucleotide positions investigated (e.g.,
a silent mutation) can be expected to affect both the wild type
probe system and the mutant-specific padlock probes, leading to
loss of both signals. This generates a distinct signal profile from
true negatives and positives, indicating the need for further
analysis of the exact genotype. The 526 TAC mutant DNA sample
yielded a significantly positive but slightly weak signal in the
mutant-specific probe cocktail. SMD control measurements
showed that this sample yielded an equal number of RCA
products as the 516 GTC and 531 TTG mutant DNA samples
(data not shown). We speculate that the particular padlock probe
sequence (526 TAC) lead to a secondary structure formation
which impeded hybridization of the oligonucleotide-conjugated
magnetic nanobeads to the RCA products. The hybridization to
the magnetic nanobeads can further be improved by adjusting
hybridization conditions or by changing padlock probe backbone
sequences.
Other readout formats in addition to the VAM-NDA could be
explored for this padlock probe assay. For example, the SMD
method used here to evaluate the probes has better sensitivity,
although it requires more sophisticated equipment in its current
form [33]. A colorimetric readout format, such as visualization by
horse radish peroxidase and 3,3,5,5-Tetramethylbenzidine,
enabling parallel readout in a microplate, is another possibility
[36]. The current LOD of the method developed here may be
considered unsatisfactory for direct testing of clinical specimens.
The sensitivity of the assay developed by Ke et al. [36] was
increased by adding another cycle of circle-to-circle amplification,
enabling a limit of detection of 600 targets per reaction. A similar
increase in sensitivity can be expected for this assay by introducing
an additional cycle of amplification to the current protocol.
Furthermore, the assay in its current design is not able to provide
information about which exact mutation is present in the target. If
desired, this can be achieved by employing a multiplexed array
readout. The assay could then also be expanded to detect
mutations in other genes associated with drug resistance in M.
tuberculosis.
Drug-resistant M. tuberculosis is a serious public health problem
that threatens progress made in TB care and control worldwide.
Drug resistance must not be ignored. Sharpened combined and
novel efforts by the international community are needed to
develop strategies to prevent further development of resistance,
and to stop transmission of already drug-resistant strains.
Molecular diagnostics can play an important part of future TB
control. We have developed a molecular method for detection of
RIF resistance in M. tuberculosis by padlock probes and a magnetic
bead-based readout. We show that padlock probes can specifically
detect mutations in the rpoB gene, and contrary to other
PCRbased mutation detection methods [37,38,39], the assay is easily
multiplexed and flexible.
Supporting Information
Figure S1 Confirmation of wild type probe system
improvements. Ten attomole synthetic DNA was used as
template. The different padlock probe systems had the following
properties. (A): Long target-hybridizing duplex part compared to
the single-stranded backbone. (B): Shortened duplex and increased
backbone. (C): The longest gap-fill oligonucleotide equipped with
a hinge. (D): B and C combined. (NC): negative control for
system D. RCA products were hybridized to fluorescent
dyecoupled oligonucleotide and visualized in a confocal microscope.
(TIF)
Figure S2 Determination of wild type probe system
limit of detection by the magnetic bead-based readout
format. Ten-fold dilutions of synthetic target representing wild
type rpoB locus was tested. Brownian relaxation frequency was
measured for each sample and to account for differences in
ironoxide content between samples, the data were normalized using
the constant value of the in-phase component of the volume
susceptibility,. NC: negative control.
(TIF)
Figure S5 Investigation of blocking effects during RCA.
Padlock probes designed for the same DNA strand tested in
singleplex (516 GTC) or duplex (531 TKG degenerated probe at
the 39-end nucleotide of padlock probe) on M. tuberculosis clinical
isolates harboring either a 516 GTC or 531 TTG mutation in
rpoB, and compared with nine-plex assay in presence of all mutant
specific padlock probes. RCA products were hybridized to
fluorescent dye-coupled oligonucleotide and visualized in a
confocal microscope. NC: negative control.
(TIF)
Figure S6 Optimization of suboptimally performing
padlock probes. The target-hybridizing arms of the original
degenerated padlock probes P5002, (rpoB 526 CKC) were
shortened to yield P5926, which performed better on both 526
CTC and 526 CGC targets.
(TIF)
Oligonucleotides used in the study.
data of the experiment presented in
Acknowledgements
Figure S4 Full frequency scan data used to determine
the normalized for each measurement presented in
Figure 5. Panels AC displays data for the wild type probe
system, cocktail of nine mutant-specific padlock probes, and M.
tuberculosis complex padlock probe respectively while panel D
displays the data for all negative control measurements.
We gratefully acknowledge Pontus Jureen and Sven Hoffner for helpful
comments on the study design and Ivan Hernandez-Neuta for valuable
comments on the manuscript.
Conceived and designed the experiments: AE MS MN DH. Performed the
experiments: AE TZ DH. Analyzed the data: AE TZ MS MN DH. Wrote
the paper: AE TZ DH.
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