Molecular Beacons: A Real-Time Polymerase Chain Reaction Assay For Detecting Salmonella
Molecular Beacons: A Real-Time Polymerase Chain Reaction Assay For Detecting Salmonella
Molecular Beacons: A Real-Time Polymerase Chain Reaction Assay For Detecting Salmonella
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To whom correspondence should be addressed. Fax: 909-787- Abbreviations used: MB, molecular beacon; CFU, colony-forming
2425. Email: Wilfred@engr.ucr.edu. unit; MWD, Los Angeles Metropolitan Water District.
DNA Extraction
One and one-half milliliters of each culture was
transferred into Eppendorff tubes and centrifuged at
1400 rpm for 2 min. The pellets were resuspended in
567 l of TE buffer. Thirty microliters of 10% SDS and
FIG. 1. (a) Molecular beacons anneal to complementary template. 3 l of 20 mg/ml proteinase K were added to each
This results in separation of the quencher from the fluorophore and sample. After thorough mixing, the samples were in-
increases fluorescence. (b) Molecular beacons do not form stable cubated in a water bath at 37°C for 1 h. After incuba-
hybrids with mismatched template sequences. The proximity of the tion, 100 l of 5 M NaCl was added and mixed
quencher to the fluorophore when the stem portion is hybridized
results in very low background fluorescence.
thoroughly. Eighty microliters of a 10% CTAB
(hexadecyltrimethyl ammonium chloride) in 0.7 M
NaCl solution was added to each sample, followed by
incubation in a water bath at 65°C for 10 min. Then 0.8
rescence is observed even in the presence of a target ml of chloroform:isoamyl alcohol (24:1) was added after
strand that contains only a single nucleotide mismatch
incubation. The samples were briefly vortexed and cen-
(7). Since unhybridized beacons do not have to be sep-
trifuged for 5 min. The aqueous supernatants were
arated, they can be included in PCR reactions, permit-
transferred into clean tubes. This was followed by a
ting the progress of the reaction to be followed in real
second extraction. Four hundred microliters of isopro-
time. In real-time PCR, MBs as well as the primers
panol was added and each sample was gently mixed
hybridize to the templates during the annealing stage.
until DNA precipitates were visible. The precipitates
Data are collected at each annealing stage, when MBs
were pelleted by centrifuging at 1400 rpm for 10 min.
are hybridized to the target. When the temperature is
The pellets were washed with 200 l of ice-cold 70%
increased during the extension stage, MBs dissociate
ethanol. After removing the ethanol, the pellets were
from the templates and do not interfere with polymer-
allowed to dry in a vacuum oven at 50°C for 5 min and
ization. As the target strands synthesized in a reaction
resuspended in 100 l of TE buffer. DNA recovery was
accumulate, the fraction of MBs that is bound to tar-
confirmed by gel electrophoresis. PCR reactions were
gets will increase, causing a brighter fluorescent sig-
carried out with each sample to confirm the DNA qual-
nal. Recent studies have successfully employed MB for
a variety of PCR applications (8 –11). ity. PCR products were verified by gel electrophoresis.
In this paper, we described the detection of Salmo- This method provided good yields of PCR quality DNA
nella by real-time PCR assay based on MB. A 122-bp and was used for subsequent DNA extractions.
region of the himA gene was used as the target. We
demonstrate that the real-time assay is highly specific Design of Molecular Beacons
with the capability to discriminate even a single base
mismatch. As few as 2 colony-forming units (CFU) can MB (BHMA1) 5⬘-FAM-CGCTATCCGGGGCGTA-
be detection with the MB-based PCR assay. ACC-CGTAGCG-3⬘-DABCYL was designed to be per-
fectly complementary to the himA gene of Salmonella.
MATERIALS AND METHODS The target sequence contained two nucleotide mis-
matches, relative to the same region in the himA gene
Bacterial Strains, Media, and Culture Conditions of E. coli.
Salmonella typhimurium LT2, Escherichia coli MBs were synthesized by MIDLAND Certified Re-
W3110, Citrobacter freundii ATCC 8090, and Enter- agent Company. BHMA1 was labeled at the 5⬘ end
obacter cloacae ATCC13047 were obtained from the with fluorescein (6-FAM) and the quencher 4-(4⬘-di-
American Type Culture Collection (ATCC). Seven Sal- methylaminophenylazo)benzoic acid (DABCYL) at the
monella isolates were obtained from the Los Angeles 3⬘ end. The stem sequence was selected so that they
County Sanitation Districts. These isolates were iden- would not complement the sequences within the loop
tified and labeled: (1) Salmonella OC1, (2) Salmonella region. The length of the beacon was selected so that
Group C-Factor 7, (3) S. choleracious, (4) S. enteriditis, the annealing temperature is slightly higher than the
(5) S. heidelberg, (6) S. newport, and (7) S. thompson. annealing temperature of the PCR primers. The bea-
All cultures were grown in Nutrient Broth (DIFCO cons were resuspended in TE buffer, stored at ⫺20°C,
Laboratories) overnight at 37°C. The cell count of over- and protected from light. Aliquots (16 mM) were pre-
night cultures was estimated by enumeration of spread pared and used for subsequent studies.
168 CHEN, MARTINEZ, AND MULCHANDANI
RESULTS
Thermal Denaturation Profiles
To explore the optimal hybridization temperature
FIG. 2. Thermal denaturation profiles of the molecular beacon.
between the beacon and the target sequence, the ther-
mal denaturation profiles of the beacon and the bea-
Thermal Denaturation Profiles
Thermal denaturation profile studies were con-
ducted to determine the optimal annealing tempera-
ture for the real-time PCR. The changes in fluorescence
of a 50 l solution containing 0.3 M of the beacon
probe with or without 0.9 M of a perfectly complemen-
tary single-stranded oligonucleotide were measured.
The samples were place in a Perkin–Elmer ABI Prism
7700 Sequence Detector System and heated to 90°C for
5 min. The temperature was then reduced at 1°C per
minute increments to 20°C. Data were recorded at each
temperature interval. The optimal hybridization tem-
peratures for each beacon were determined from these
plots.
PCR Primers
Primers SHIMAF 5⬘-CGTGCTCTGGAAAACGGT-
GAG-3⬘ and SHIMAR 5⬘-CGTGCTGTAATAGGAA-
TATCTTCA-3⬘, specific for a 122-bp fragment of the
himA gene, were previously reported by Bej (6). To
amplify the himA region of E. coli, a similar pair of
primers (EHIMA-forward, 5⬘-CGCGCTCTGGAAAA-
CGG-3⬘; and EHIMA-reverse, 5⬘-CGTGCTGTAATGG-
GAATATC-3⬘) were designed. The primers used were
synthesized by GENOSYS. Primers were resuspended
in TE buffer and stored at ⫺20°C. Aliquots (20 mM) of
each primer were prepared and used for PCR. FIG. 3. (a) Real-time PCR. Amplification of the Salmonella himA
gene, using 10-fold serial dilutions of template DNA. These experiments
focused on the detection of low concentrations of Salmonella DNA
PCR Conditions ranging from approximately 18,000 to 1.8 copies. Symbols: ■, 18,000
The Perkin–Elmer ABI Prism 7700 sequence Detec- CFU/PCR; E, 1800 CFU/PCR; }, 180 CFU/PCR; F, 18 CFU/PCR; ⫹, 2
CFU/PCR; ⫺, no template; *, buffer. (b) Gel electrophoresis. PCR reac-
tion System was used for real-time analyses. Thermal tions using 10-fold dilutions of template DNA: (1) 180,000,000 CFU/
cycling conditions were specified as follows: initial PCR, (2) 18,000,000, (3) 1,800,000, (4) 180,000, (5) 18,000, (6) 1800, (7)
melting at 96°C for 10 min, followed by 40 cycles of 180, (8) 18, (9) 2, (10) no template control, (11) DNA ladder.
REAL-TIME POLYMERASE CHAIN REACTION ASSAY FOR DETECTING Salmonella 169
FIG. 4. Critical cycle. The critical cycles for PCR samples containing 10-fold dilutions of template DNA were plotted against the DNA
concentration (cells/PCR). The results showed a linear relationship between these values, which can be used to develop a standard
quantification curve.
con–target hybrid were determined using a PE/ABI normalized fluorescent measurement vs the PCR cycle.
7700 fluorescence reader. At lower temperatures per- This plot clearly shows the progression of the amplifi-
fectly complementary beacon–target hybrids fluo- cation reaction in each sample. As few as 2 CFU could
resced brightly. As the temperature was raised, a point be detected by the real-time PCR assay (Fig. 3a). Re-
was reached at which the hybrids dissociated (Fig. 2). sults were also verified by gel electrophoresis (Fig. 3b).
This was accompanied by a marked decrease in fluo- Although the intensity of the amplified fragments cor-
rescence. Conversely, the beacons unravel at higher related to the initial amount of template DNA, the
temperature and exhibited a melting temperature visual detection limit was noticeably lower at approx-
around 62°C. In the temperature interval from 50 to imately 180 CFU.
62°C, the probe–target hybrids elicited significantly The critical cycle (C t), defined as the cycle at which a
stronger fluorescence than the probe alone—thus al- significant increase in fluorescence is first recorded,
lowing the detection of target sequence at these tem- increases as the initial number of template molecules
peratures. Initial studies with real-time PCR were con- DNA decreases (12). This was expected because sam-
ducted between this temperature range and the ples containing low concentrations of template DNA
highest sensitivity was obtained at 57°C. This temper- would require more PCR cycles to replicate enough
ature was chosen as the annealing temperature for copies to produce a significant fluorescent signal. Thus,
subsequent real-time PCR studies.
C t can potentially be used to quantify the input target
concentration. For our real-time PCR assays, the C t
Real-Time PCR Analysis values decrease linearly with increasing target quan-
The ability of MB to detect Salmonella in real-time tity up to 18,000 CFU (Fig. 4). At higher initial target
PCR assays was investigated. Different initial concen- concentrations, the endpoint plateau at a lower fluo-
trations of template DNA isolated from S. typhi- rescent value than would be expected. This phenome-
murium were used. DNA extracts were diluted with non has been suggested to be attributable to late cycle
water, in 10-fold serial dilutions. Five microliters of inhibition (12).
template solution was added to each 50 l of PCR The variability between different sample prepara-
reaction. Colony-forming unit was used to represent tions was investigated. Five different sets of real-time
the initial template concentrations. A no-template-con- PCR assay were performed with initial target ranging
trol, in which sterile buffer was substituted for tem- from 1 to 18,000 CFU. Each amplification was per-
plate DNA, was used in each experiment. This control formed in duplicate. Comparison of C t values for each
was used to subtract any fluorescence that is not di- duplicate sample showed minimal variation, indicating
rectly related to amplification. Figure 3a shows the that the PCR amplification is highly reproducible.
170 CHEN, MARTINEZ, AND MULCHANDANI
TABLE 1
Variation in Real-Time PCR Assay
Ct
Comparison of C t values of the five different sets of nella species using the SHIMA primers and a Salmo-
assay also revealed little variability (Table 1). More nella-specific DNA probe showed negative results for
importantly, the rate of fluorescent change at each all species tested except for C. freundii and E. cloacae,
target concentration was similar among the five differ- which produced false-positive results from both the
ent assays. primers and the probe (R. DeLeon, personal communi-
cation). To confirm the specificity of the beacons, DNA
Specificity of the Real-Time Assay isolated from C. freundii and E. cloacae were obtained
from MWD and subjected to the real-time PCR assay.
To demonstrate the beacon’s specificity for Salmo- DNA bands of 122 bp were detected for both C. freundii
nella, primers were designed to specifically amplify the and E. cloacae (Fig. 6a). However, neither one of these
same region of the himA gene in E. coli. PCR products organisms was detected by the beacons (Fig. 6b).
were examined through gel electrophoresis. A 122-bp The ability of the beacons to target other Salmonella
product was amplified from both the E. coli and Sal- species rather than S. typhimurium was tested. Seven
monella templates. However, real-time PCR studies different Salmonella species isolated by the LA County
showed that the MB, specific for Salmonella, failed to Sanitation District were analyzed by the real-time
detect the E. coli product (Fig. 5). Because there are PCR assay. All seven targets produced strong fluores-
two base pair mismatches within the E. coli amplicons, cent response (⌬R n ⬎ 0.5 after 40 cycles). The pres-
the MBs form unstable hybrids that dissociate dur-
ence of the PCR-amplified himA targets was also con-
ing the annealing stage, at which fluorescence is re-
firmed by gel electrophoresis (data not shown).
corded—thus demonstrating the ability of MB to dis-
criminate between very similar sequences.
A recent PCR study conducted by the Los Angeles DISCUSSION
Metropolitan Water District (MWD) on 50 non-Salmo-
The MB-based PCR assay provides the possibility of
real-time quantitatively detection of specific target di-
rectly in the PCR tube. The reported assay could detect
as few as 2 CFU of S. typhimurium and also cover a
wide dynamic range of detection, all in a real-time
manner. Detection of Salmonella using MB is also ex-
traordinarily specific. Even in the presence of a similar
himA sequence from E. coli, no increase in fluorescence
was detected. Perhaps the most powerful aspects of MB
is the capability to distinguish false-positive results
from PCR amplification. For example, a 122-bp ampli-
con was detected from both C. freundii and E. cloacae
using the Salmonella primers. Even the use of a linear
hybridization probe failed to provide the correct iden-
tity. However, no response was produced from both
strains in the real-time PCR assay, again demonstrat-
FIG. 5. Target specificity. Real-time PCR amplification of E. coli
and Salmonella himA genes. Beacons specific for Salmonella failed ing the highly specific nature of MB.
to detect the himA homologue in E. coli. Symbols: E, E. coli; Œ, Analysis of fluorescence data recorded at each an-
Salmonella; ⫺, no template control. nealing stage gives a clear profile of the amplification
REAL-TIME POLYMERASE CHAIN REACTION ASSAY FOR DETECTING Salmonella 171
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