JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2002, p. 4143–4147
0095-1137/02/$04.00⫹0 DOI: 10.1128/JCM.40.11.4143–4147.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 40, No. 11
Rapid and Specific Detection of Mycobacterium tuberculosis from
Acid-Fast Bacillus Smear-Positive Respiratory Specimens and
BacT/ALERT MP Culture Bottles by Using Fluorogenic
Probes and Real-Time PCR
Nancimae Miller,1* Tim Cleary,1,2 Günter Kraus,2 Andrea K. Young,2 Gina Spruill,2
and H. James Hnatyszyn2
Department of Pathology1 and Department of Microbiology and Immunology,2 University of Miami
School of Medicine, Miami, Florida 33136
Received 1 July 2002/Returned for modification 6 August 2002/Accepted 29 August 2002
Mycobacterium tuberculosis remains a serious public health
issue due to its high risk of person-to-person transmission,
morbidity, and mortality (15, 20). Currently, approximately 8
million new infections and 3 million deaths are attributed to
tuberculosis (TB) each year (L. B. Reichman, Letter, Chest
112:855, 1997). Progressive increases in TB infections are expected, and a worldwide annual incidence of 12 million cases
by 2005 is predicted by the World Health Organization (20).
The resurgence of TB in industrialized countries since the
mid-1980s, primarily due to the increased incidence of immunocompromised patients with AIDS, and the emergence of
multidrug-resistant strains of M. tuberculosis has accented the
need for rapid diagnosis of this disease (15, 20). Rapid detection of active TB infection is critical for effective patient management and implementation of infection control measures.
Conventional detection of mycobacteria is based on a number of protocols, including microscopic examination of smears
stained with the Ziehl-Neelsen stain or auramine fluorescent
dye and selective culture techniques (8, 17, 26, 39). The key
aspect of TB control is rapid diagnosis, which for many years
has been based on the staining of smears for the presence of
acid-fast bacilli (AFB). The AFB smear test lacks specificity, so
there is a need for a laboratory test for specific detection of the
M. tuberculosis complex (MTB) that can be performed within a
short period of time.
Molecular methods, such as DNA probes and nucleic acid
amplification tests, offer a rapid, specific, and sensitive approach to the detection of MTB from liquid cultures (29, 35)
and for detection of TB directly from clinical specimens (1, 9,
11, 16, 22, 32, 37, 38). Nucleic acid amplification methods have
been applied in the clinical laboratory with great success; however, these procedures are often labor intensive, and the FDAapproved nucleic acid amplification-based assays for MTB displayed high specificity but variable sensitivity (6, 7, 25).
Multiple steps are required in the amplification and detection
steps involving user manipulations at each point of the assay
that have the potential for error and sample contamination.
Real-time PCR techniques, involving fluorescent dyes or
fluorophores with a spectrofluorometric thermal cycler, have
been used to develop a number of rapid and sensitive assays for
identification of bacteria and viruses, including herpes simplex
virus (12, 14), varicella-zoster virus (13), cytomegalovirus (33),
Legionella pneumophila (2), enterohemorrhagic Escherichia
coli (3), Staphylococcus aureus (28), Mycobacterium bovis (34)
and also for genes conferring drug resistance in Helicobacter
pylori (21), hepatitis B virus (5), S. aureus (28) and M. tuberculosis (36). Fluorogenic probes, including molecular beacons
and paired hybridization probes, can be designed to recognize
a specific sequence from a target gene and can enhance the
specificity and sensitivity of the assay over those of conven-
* Corresponding author. Mailing address: Department of Pathology
(D33), 1611 NW 12 Ave., Holtz Tower 2115, Miami, FL 33136. Phone:
(305) 585-6258. Fax: (305) 585-0008. E-mail: nmiller@med.miami.edu.
4143
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A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium
tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive
respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC
PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium
genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The
results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test.
Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB
was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive
by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms;
both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and
moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB
smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested
negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive
by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This
real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample
available for testing.
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MILLER ET AL.
tional PCR techniques. Real-time fluorescence has been used
to quantitate MTB DNA in sputum during treatment of TB
patients using the TaqMan system (10). We recently reported
a rapid and sensitive method for the identification of MTB by
amplification of the internal transcribed spacer (ITS) and specific fluorogenic probes for MTB in the LightCycler (LC) system (19).
In this clinical laboratory-based study, specific identification
of MTB was shown by using the LC PCR system (Roche
Molecular Biochemicals) and real-time detection with specific
fluorogenic probes. We were able to detect MTB DNA in
processed AFB smear-positive respiratory specimens and MP
culture bottles.
Respiratory specimen processing and identification of mycobacteria. Respiratory specimens submitted for culture were liquified and decontaminated with
N-acetyl cysteine–2.5% NaOH and concentrated by centrifugation (27). The
sediment was used to inoculate a selective 7H11 agar plate and a supplemented
MP culture bottle (Organon Teknika, Durham, N.C.) and to prepare two smears
for staining. The respiratory, fixed smears were stained with an auramine fluorochrome stain (18). The number of fluorescent AFB was reported on the basis
of the following criteria used in our laboratory (at a magnification of ⫻400): no
AFB seen ⫽ negative; one to three per slide ⫽ 1⫹ (rare); one to nine per 10
fields ⫽ 2⫹ (few); one to nine per field ⫽ 3⫹ (moderate); and more than nine
per field ⫽ 4⫹ (many) (24).
MP culture bottles were incubated at 35°C in 5% CO2 and monitored for
growth for 6 weeks by using the BacT/ALERT 3D instrument according to
manufacturer’s instructions. When growth was detected, a smear was prepared to
confirm the presence of acid-fast organisms and the liquid medium was subcultured onto blood agar and a 7H11 plate. Isolates of mycobacteria growing on
solid medium were identified by DNA probes (Accuprobe; Gen-Probe, Inc., San
Diego, Calif.) for M. tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae, and Mycobacterium kansasii or by conventional
biochemical tests performed according to standard protocols (18, 23).
DNA extraction for PCR protocols. Respiratory specimens that were AFB
positive were prepared for PCR using the Roche sputum preparation kit (Roche
Diagnostics, Indianapolis, Ind.). The remaining lysates were frozen at ⫺20°C for
use in the real-time PCR assay. Culture bottles that were AFB positive were
prepared for PCR by using a Chelex 100 resin protocol. MP culture fluid (0.5 ml)
was concentrated by centrifugation at 15,000 ⫻ g for 15 min in a 1.5-ml screw-cap
microcentrifuge tube. The supernatant was removed, and the pellet was resuspended in 100 l of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]). This
suspension was then transferred to a 1.5-ml tube containing 100 l of a 40%
solution of Chelex 100 resin (32) (Sigma Chemical, St. Louis, Mo.) in 10 mM
Tris-HCl (pH 8.0) containing 0.2% laureth-12 (PPG industries, Gurnee, Ill.).
The sample was resuspended by vortexing and incubated at 56°C for 30 min
followed by incubation at 100°C for 10 min. The sample was vortexed again,
allowed to cool, and centrifuged for 10 min to clarify the supernatant, which was
transferred to another tube for storage prior to evaluation using real-time PCR.
AMPLICOR M. tuberculosis PCR test. AFB smear-positive respiratory specimens were processed for PCR directly from the decontaminated, concentrated
sediment according to the package insert for the Amplicor M. tuberculosis test
(Roche Diagnostics), as previously described (9). All manipulations of positive
smear specimens were performed in a biological safety cabinet. PCR amplification and detection were performed according to manufacturer’s guidelines. For
this assay, 50 l of the lysate was used for amplification in a total volume of 100
l.
LightCycler real-time PCR for the ITS sequence. Oligonucleotides designated
Sp1 (5⬘-ACCTCCTTTCTAAGGAGCACC-3⬘) and Sp2 (5⬘-GATGCTCG
CAACCACTATCCA-3⬘) were used to amplify an approximately 220-bp fragment of the ITS sequence (EMBL accession number L15623) from Mycobacterium (30, 31). Amplified product was detected either by the use of SYBR Green
I dye or by using specific fluorescent probes. The SYBR Green I dye allows
detection of any double-stranded DNA generated during PCR; both specific and
nonspecific products will generate a signal. In order to specifically identify MTB,
paired fluorogenic hybridization probes were designed to recognize a region in
the ITS fragment (19). The 5⬘ anchor probe was modified at the 3⬘ end with the
TABLE 1. Detection of M. tuberculosis DNA from acid-fast smear
positive respiratory samples by real-time PCR and Amplicor PCR
No. of samples with acid-fast smear resulta
Real-time PCR/
Amplicor PCR
1⫹
2⫹
3⫹
4⫹
Total
Positive/positive
Negative/positive
Positive/negative
29
2
1
30
0
0
19
0
1
23
0
0
101
2
2
a
Fluorescence microscopy (magnification, ⫻400). 1⫹, one to three per slide;
2⫹, one to nine per 10 fields; 3⫹, one to nine per field; 4⫹, more than nine per
field.
donor fluorophore, 6-carboxy-fluoroscein, and designated 4602 (5⬘-GTGGGGC
GTAGGCCGTGAGGGGTTC-FAM-3⬘). The 3⬘ detection probe was designated 4600 (5⬘-LC640-GTCTGTAGTGGGCGAGAGCCGGGTGC-ⴱ-3⬘) and
was designed to hybridize 3 bp downstream of the anchor probe. The 3⬘ end of
the probe was also phosphorylated (as indicated by the asterisk) to prevent
amplification and extension with the 3⬘ SP2 primer. The hybridization probes
were synthesized by Synthegen LLC (Houston, Texas).
For each sample, 2 l of template DNA was incorporated into a 10-l PCR
containing the amplification oligonucleotides and MTB-ITS hybridization probes
using the LightCycler DNA Master Hybridization kit (Roche Biochemicals). The
optimized LC PCR protocol included an initial denaturation step at 95°C for 30 s
and was followed by a touchdown PCR protocol using the following conditions:
95°C for 0 s, 61°C for 15 s, and 72°C for 30 s for five cycles; 95°C for 0 s, 60°C for
15 s, and 72°C for 30 s for 5 cycles; 95°C for 0 s, 59°C for 15 s, and 72°C for 30 s
for five cycles; and 95°C for 0 s, 59°C for 5 s, and 72°C for 30 s for 35 cycles. The
total time of amplification, detection, and analysis using this optimized protocol
is approximately 40 to 45 min for 32 samples. Fluorescence measurements are
made in every cycle. The threshold cycle (Ct) value is the cycle at which there is
a significant increase in fluorescence, and this value is associated with an exponential growth of PCR product during the log-linear phase. Positive and negative
controls were used that had been prepared by cloning the amplified ITS region
into a pGEM vector from M. tuberculosis (pGEM MTB) and M. kansasii (pGEM
MK), respectively (19). The total time for amplification, detection, and analysis
is approximately 40 min for up to 32 samples per run.
RESULTS
Real-time MTB detection from concentrated respiratory
specimens. Residual DNA lysates prepared for Amplicor MTB
PCR from respiratory specimens that were AFB smear positive
were tested in the LC PCR assay using 2 l of the lysate (Table
1). We tested a total of 135 smear-positive specimens. There
were 105 AFB smear-positive specimens tested that grew
MTB. Of this group, 103 specimens were positive by LC PCR
and Amplicor PCR. The two samples that were negative by LC
PCR both contained low numbers of organisms by acid-fast
smear and were negative on repeat testing. The two samples
that were negative in the Amplicor PCR assay contained rare
and moderate numbers of organisms by acid-fast smear, and
both were positive in the LC assay. These samples were not
tested further in the Amplicor PCR test, since both patients
had other specimens that were positive in both PCR assays.
There were 30 AFB smear-positive specimens tested that grew
mycobacteria other than MTB, and all were negative in the LC
PCR and Amplicor PCR assays. The sensitivity and specificity
of the LC PCR assay for the AFB smear-positive specimens
was 98.1% (103 of 105) and 100% (30 of 30), respectively.
The Ct, which is a measure of the cycle of detection of
product, correlated with the acid-fast smear result. The average Ct value for 4⫹, 3⫹, 2⫹, and 1⫹ samples was 28.4, 30.4,
32.4, and 36.0 cycles, respectively.
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MATERIALS AND METHODS
J. CLIN. MICROBIOL.
VOL. 40, 2002
PCR ASSAY FOR MTB DETECTION
TABLE 2. Detection of M. tuberculosis DNA from BacT/ALERT
MP (MP) culture bottles by real-time PCR versus organism cultured
from bottle
Organism cultured
a
No.
positive
No.
negative
114
0
0
0
0
0
0
0
0
0
0
0
67
18
12
6
5
2
2
1
2
2
No. of
patients
41
43
8
9
5
4
2
2
1
1
2
a
All are of the genus Mycobacterium. MOTT, mycobacteria other than tuberculosis.
Specific detection of MTB from MP culture specimens. We
tested 231 MP cultures from respiratory specimens obtained
from 118 patients. Of these, 114 were positive by LC PCR and
all were culture positive for M. tuberculosis (Table 2). These
samples were obtained from 41 patients. The remaining 117
specimens were negative in the LC assay. To examine for
possible sample inhibition of amplification, all negatives samples were amplified using SYBR Green I, a fluorochrome that
binds nonspecifically to double-stranded DNA. A melt curve
analysis performed on the LC confirmed the presence of ITS
fragment amplification in all specimens. All of these specimens
were culture positive for various Mycobacteria species. At least
eight different species were identified from these specimens.
As expected for our institution, M. avium or M. intracellulare
(MAI) and M. kansasii accounted for the majority of these
isolates. The sensitivity and specificity of the LC PCR assay for
the MP culture bottles specimens was 100% (114 of 114) and
100% (117 of 117), respectively.
The pGEM MTB plasmid was positive in all assay runs, and
the pGEM MK plasmid and water negative controls were negative throughout these experiments. The average Ct value for
the pGEM MTB plasmid was 23.2 (range of 18.7 to 25.4) over
a 10-week period.
DISCUSSION
Due to the slow growth of M. tuberculosis, rapid identification methods using molecular techniques have been developed
and utilized in the clinical laboratory. However, these methods
require many manipulations and take several hours to complete. The LC instrument is a commercially available system
designed to decrease the time of the PCR by monitoring the
amplification of the target sequences in real time by fluorescent probes. This technology is a significant breakthrough in
PCR amplification and amplicon detection compared to conventional detection methods, and the benefits for clinical assays have been reported (12, 13, 14, 33, 40).
Previously we utilized the LC and ITS oligonucleotide primers and probes on a collection of 20 different ATCC strains to
determine the sensitivity and specificity of the assay (19).
These strains represented the most commonly isolated species
that are recovered in the clinical mycobacteriology laboratory.
When the SYBR Green I dye was used as the fluorophore, we
were unable to specifically identify MTB from among other
Mycobacterium species. Several of the mycobacteria species
produced amplified ITS products that had very unique melting
points; however, most melting point profiles were too similar to
reliably distinguish these species. The specific fluorescence resonance energy transfer probes we designed were shown to
detect the MTB ITS sequence, while all other mycobacteria
species tested negative. A plasmid containing the ITS sequence
was prepared for MTB (pGEM MTB) and M. kansasii (pGEM
MK). When the assay was performed using the pGEM MTB
plasmid as the DNA template, we were consistently able to
detect less than 200 fg per PCR. The pGEM MK plasmid,
which was tested at a concentration of 2,000 pg per PCR, was
always negative.
In this study, the LC real-time assay was evaluated for the
specific identification of MTB from AFB smear-positive clinical specimens and from MP culture bottles. The LC assay was
positive for 103 of 105 (98.1%) specimens that were culture
positive for MTB. The two negative MTB samples both contained rare numbers of organisms by acid-fast smear and were
submitted from two patients. For one of the patients, this
specimen represented the only AFB smear-positive sample
from several specimens that were submitted for this patient.
All of the patient’s cultures grew MTB. The other patient had
two additional respiratory samples that were AFB smear positive; both of these samples were positive by the LC assay.
Therefore, according to our clinical pathway for the diagnosis
of TB in the hospital, this patient would have been correctly
identified as positive for TB. We were impressed with the
ability of the LC assay to detect positive patients because the
sample size in this assay was much smaller than that in the
Amplicor PCR assay. The latter test uses 50 l of the lysed
sediment, while the LC assay used only 2 l of sample. Also,
we did see a clustering of the Ct values that correlated with the
acid-fast smear result. The greater the number of organisms
present on the smear, the lower the Ct value for that specimen
was. We have not evaluated this assay using AFB smear-negative samples. It is our feeling that these samples would require
purification and concentration of the crude lysate using a silica
membrane such as the QIAamp DNA mini kit (Qiagen, Valencia, Calif.). This method has been used to remove inhibitors
in clinical specimens extracted by using the Amplicor protocol
(4). In fact, we tested one acid-fast culture from a stool specimen and it was negative, possibly from inhibition. This specimen was from a patient who had respiratory samples that were
positive in the LC assay.
The LC protocol proved very useful for the quick identification of MTB from MP culture bottles. Of the 231 liquid
cultures that were tested in this study, 114 samples grew MTB
and all were positive in the LC assay. The remaining 117
specimens were negative in the LC assay. The organisms isolated from these bottles included those that are commonly
found in the clinical laboratory. MAI and M. kansasii accounted for 74% of these species.
In the LC assay, detection of amplified nucleic acid products
is accomplished in a closed system; the capillary reaction vessels are never opened after the cycling process has started.
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M. tuberculosis
MAI
M. kansasii
M. fortuitum
M. gordonae
M. chelonae
M. asiaticum
M. nonchromogenicum
M. simiae
M. kansasii/MAI
MOTT
Result with real-time
PCR
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MILLER ET AL.
ACKNOWLEDGMENTS
This work was funded by the Sylvester Comprehensive Cancer Center and the Department of Pathology.
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Therefore, there is no opportunity for carryover contamination
to occur postamplification. There are steps in the extraction
and processing procedures that may be susceptible to crosscontamination of target nucleic acid between specimens. To
address this potential problem, specimen extraction would be
performed in a biologic safety cabinet, and the sample loading
should be done in a separate PCR workstation that is decontaminated with UV light after each use.
This LC assay proved to be very quick and potentially laborsaving in the laboratory. The time required for DNA extraction
was 1.5 h, followed by 10 min for pipetting and 30 min for
cycling and detection of amplified product. No other manual
manipulations are necessary after the capillary reaction vessel
was placed in the LC instrument. In contrast, the Amplicor M.
tuberculosis PCR test took more than 5 h and required additional manual manipulation throughout the procedure. One
hour was required for DNA extraction, 20 min was required for
reagent preparation and pipetting of amplification mix, 1.8 h
was required for amplification, and 2 h was required for detection by colorimetric microwell plate probe hybridization.
Our goal is to implement this LC PCR method into our diagnostic laboratory for routine detection of MTB from AFB
smear-positive specimens and MP cultures. Also, we would like
to validate a protocol that we could use on AFB smear-negative specimens. Potentially, the combination of real-time PCR
with sequence-specific fluorogenic probes can be optimized to
detect mycobacterial DNA or RNA from sputum, bronchoalveolar lavage, blood, cerebrospinal fluid, pleural fluid, or tissue
samples.
J. CLIN. MICROBIOL.
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