University of Helsinki, Faculty of Veterinary Medicine, Department of Basic Veterinary Sciences, Section of Microbiology, PO Box 57, FIN-00014 Helsinki University, Finland
Correspondence
Airi Palva
Airi.Palva{at}Helsinki.fi
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ABSTRACT |
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Abbreviations: GI, gastrointestinal
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INTRODUCTION |
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Without a doubt, the available microbiological methods provide valuable
information on the faecal microbial composition. However, they are not optimal
for monitoring quantitative changes, especially those of subdominant microbial
species or groups. Therefore, we used real-time PCR with TaqMan probes (5'-nuclease
assay) or SYBR Green I for the specific detection of selected bacteria
from faecal DNA. Real-time PCR is based on the continuous monitoring of changes
in fluorescence during PCR and, in contrast to the conventional end-point
detection PCR, quantification occurs during the exponential phase of amplification.
Thus, the bias often observed in the PCR template-to-product ratios (Suzuki & Giovannoni, 1996) is
avoided. The TaqMan assay is based on measuring the fluorescence
released during PCR as the 5'-nuclease activity of Taq DNA
polymerase cleaves a dual-labelled fluorescent hybridization probe, designed
to bind inside the amplified region, during primer extension (Heid et al., 1996
). SYBR Green
I is an intercalating dye that gives a fluorescent signal when bound to double-stranded
DNA, while being otherwise virtually non-fluorescent. SYBR Green I lacks the
specificity of the TaqMan assay, where the fluorescent signal is
derived from a specific probe. However, SYBR Green I can be applied to the
fluorescent monitoring of any amplification reaction, which gives more flexibility
to the real-time PCR.
In this study, we designed specific rDNA-targeted oligonucleotide primers
or probes for six bacterial species or groups and applied them to the detection
and quantification of target bacteria from faecal DNA with the two real-time
PCR applications discussed above and rDNA-targeted dot-blot hybridization.
Five faecal target bacteria were chosen for the assays. Bacteroides fragilis represented the Bacteroides group and Ruminococcus productus represented the Clostridium coccoides group; these two groups
have been reported to be abundant in human faeces (Sghir et al., 2000). Bifidobacterium
longum, one of the most common bifidobacterial species in human faecal
samples (Mangin et al., 1994
; Matsuki et al., 1999
; Malinen et al., 2002
), was also chosen for the assay. Although enterobacteria
and lactobacilli form only a minor part of the GI microflora (Sghir et al., 2000
), Escherichia coli and Lactobacillus acidophilus assays were also
included. The remaining target species, Bifidobacterium lactis, does
not belong to the human GI microflora but is increasingly used in the dairy
industry as a probiotic bacterium.
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METHODS |
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Extraction and purification of DNA from faeces and pure cultures.
Bacterial DNA was isolated from pure cultures of the reference strains
and from faecal samples obtained from healthy volunteers. Removal of undigested
particles from the faeces was performed by washing the samples with repeated
low-speed centrifugations, followed by collection of the bacteria from combined
wash supernatants with high-speed centrifugation (Apajalahti et al., 1998). Cell lysis
and DNA extraction from faecal bacterial pellets and pure cultures were accomplished
with a combination of physical, chemical and enzymic steps as described by Apajalahti et al. (1998)
.
DNA concentrations were measured with a Versafluor fluorometer (Bio-Rad).
This method is based on the quantification of the intensity of Hoechst 33258
dye (bisBenzimide H 33258), which is fluorescent when bound to double-stranded
DNA, with an excitation wavelength of 360 nm and an emission wavelength
of 460 nm. Due to the high sensitivity of Hoechst 33258, the amount
of DNA required for concentration measurements is reduced and a linear quantification
of double-stranded DNA can be obtained in the range of 20 ng to 10 mg.
Design of oligonucleotide primers and probes.
Hybridization probes and real-time PCR primers were designed for six bacterial
species or groups, representing faecal microbes or bacteria used in the dairy
industry. The on-line Internet tools CLUSTAL W (Thompson et al., 1994) and FASTA3 (Pearson & Lipman,
1988
) provided by the European Bioinformatics Institute (http://www.ebi.ac.uk) as well as the PROBE MATCH and HIERARCHY
BROWSER interfaces provided by the Ribosomal Database Project (Maidak et al., 2001
; http://rdp.cme.msu.edu/html/) were utilized for identifying the primers and probes that
were able to bind with the desired specificity to the rDNA of the selected
target bacteria. The Bacteroides fragilis and Bifidobacterium
lactis assays were designed to be species-specific, whereas the remaining
four assays were set to detect closely related bacterial species. According
to the sequence analyses, the Bifidobacterium longum assay detected Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium
pseudolongum and Bifidobacterium suis. A recent study by Sakata et al. (2002)
has suggested the unification of the species Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis into
a single species. The E. coli assay targeted the Escherichia subgroup, composed of E. coli, Hafnia alvei and Shigella spp. Target bacteria for the L. acidophilus assay were L. acidophilus, Lactobacillus amylovorus, Lactobacillus
amylolyticus, Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus johnsonii. The R. productus assay
was specific for R. productus and C. coccoides. Base sequences
and modifications of the oligonucleotides used in dot-blot hybridization and
real-time PCR are shown in Tables 1
and 2, respectively. Whenever possible, dot-blot hybridization was performed
with one of the PCR primers used in real-time PCR detection of the same target
species or group. Specificity of the real-time PCR was obtained with one or
two primers selective for the intended target(s) while the TaqMan probes used had broader ranges of target sequences and could therefore
be used in conjunction with various primer sets.
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Stringent washing temperatures (Ti) for the
oligonucleotides were determined as the half-denaturation temperature by testing
temperatures at 2·5 °C intervals ranging from calculated Tm -10 °C to Tm +10 °C,
and 100 °C using the equation Tm=81·5 °C+16·6 °Cxlog10[Na+]+0·41x(mol%
G+C)-600/N, where N is the number of
nucleotides and the sodium ion concentration [Na+]
is 0·39 M (Sambrook et al., 1989).
The dot-blot hybridization was performed in the following way. DNA samples were denatured using alkali and heat and were blotted onto a positively charged nylon membrane (Boehringer Mannheim). Pre-hybridization at 65 °C for 1 h in a buffer containing 5xDenhart's solution, 5xSSC, 0·5 % SDS and 100 µg denatured Herring Sperm DNA ml-1 (Sigma) was followed by the addition of the 5'-end labelled oligonucleotide probe to a final concentration of 7x10-12 mol ml-1. The hybridization was continued for 15 min at 65 °C, after which the hybridization oven was set to 30 °C and the hybridization was continued with declining temperature until the hybridization temperature had been under Ti for 2 h. The membranes were washed with a buffer containing 2xSSC and 0·5 % SDS, four times at room temperature (5 min) and once at Ti (15 min), followed by a rinse at room temperature. The washed membranes were wrapped in heat-sealed plastic bags and the dots were quantified using the Multi-Imager with IMAGING SCREEN BI and QUANTITY ONE software (Bio-Rad).
Real-time PCR.
Performance and optimal annealing temperatures of the PCR primers (Table 2) were first tested with gradient PCR (Peltier
Thermal Cycler PTC-200; MJ Research). The standard reaction mixture consisted
of 10 mM Tris/HCl (pH 8·8), 150 mM
KCl, 0·1 % Triton X-100, 1·5 mM MgCl2, 200 µM each dNTP, 1 µM each primer and 0·03 U
Dynazyme Polymerase II µl-1 (Finnzymes).
The following PCR conditions were used. An initial DNA denaturation step at
95 °C for 5 min was followed by 30 cycles of denaturation
at 95 °C for 30 s, primer annealing at 5070 °C
for 20 s and primer extension at 72 °C for 45 s,
with a final extension step at 72 °C for 5 min. The specificity
of the primers was confirmed against the non-target test bacteria; otherwise,
results from the sequence searches against prokaryotic DNA databases were
relied upon. PCR products were analysed on agarose gels by electrophoresis.
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Optimal concentrations for various reaction components were tested for each primer set and chemistry with a dilution series of genomic DNA from the target test species. For the SYBR Green I chemistry, the effect of the polymerase type on PCR product formation was tested with a standard polymerase, Dynazyme II (Finnzymes), and the hot-start polymerases AmpliTaq Gold DNA Polymerase (Applied Biosystems), BlueTaq (Euroclone) and FastStart Taq DNA Polymerase (Roche).
The TaqMan assays were performed successfully with Dynazyme II;
therefore, switching to a hot-start enzyme was not considered. Optimal reaction
components and their concentrations for each PCR assay and chemistry (a
total of 12 different reaction mixtures) are summarized in Table 3.
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The reaction mixtures of the 5'-nuclease (TaqMan) assays consisted of 10 mM Tris/HCl (pH 8·8), 150 mM KCl, 0·1 % Triton X-100, 1·56·0 mM MgCl2, 200300 µM each dNTP, 1 µM each primer, 80400 nM fluorescent probe, 0·0240·036 U Dynazyme II µl-1 and either 10 µl of template or water. The following thermal cycling parameters were used. An initial DNA denaturation at 95 °C for 4 min was followed by 3040 cycles of denaturation at 95 °C for 15 s and a combined incubation step for primer annealing and extension at 5860 °C, during which the fluorescent signal was measured.
Quantification of target bacterial DNA in mixed DNA samples.
Specificity and sensitivity of the dot-blot hybridization probes were studied
by using 0, 10, 30, 90, 270 and 810 ng of the target strain genomic
DNA for hybridization and by using the same amounts of target DNA mixed in
500 and 1000 ng of faecal DNA. Pooled DNA samples (1350 ng)
from the genomic DNA of the non-target bacteria were used as negative controls.
Faecal DNA samples (500 and 1000 ng) without added DNA were
used for quantification of the target bacteria present in faeces.
In the real-time PCR assays, 20 ng of pooled DNA from non-target test bacteria or 20 ng of faecal DNA was applied in the PCRs together with a 10-fold dilution series of between 10 ng and 0·1 pg (approx. 25x106 to 1050 target genomes) of genomic DNA from the target species. Faecal DNA samples (20 ng) without added DNA were used for quantification of the target bacteria present in faeces.
Additionally, real-time PCR was used for the quantification of pure cultures of bacterial cells that had been introduced into faecal samples. Bacterial densities in the pure cultures were estimated either by viable counts or by microscopy, after which replicate faecal samples were spiked with 109, 108, 107, 106, 105 or 0 pure cultured cells of each test species used. Collection of bacteria, lysis of the cells and isolation of DNA were preformed as described in Extraction and purification of DNA from faeces and pure cultures.
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RESULTS |
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Results from dot-blot hybridizations with dilution series from the genomic
DNA of test target species are shown in Table 4. The expected specificity was obtained but the sensitivity
of the test was rather low (Table 4
).
With the Bifidobacterium lactis, Bifidobacterium longum, L. acidophilus and R. productus probes, 30 ng of genomic
DNA extracted from the target species were required for the generation of
a hybridization signal that was distinguishable from the background signal.
This corresponds to the application of approximately 107 target
genomes to the hybridization. With the E. coli and Bacteroides
fragilis probes, 90 and 270 ng, respectively, of genomic DNA were
required for the generation of the positive hybridization signal (Table 4
).
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Applicability of dot-blot hybridization for the detection of rDNA from
faeces
To test whether target DNA could be reliably detected in DNA of faecal
origin by dot-blot hybridization, reconstruction experiments were carried
out. Analyses of artificial DNA mixtures were performed by adding 10, 30,
90 and 270 ng of target DNA to 500 and 1000 ng faecal DNA. Hybridization
results from mixed DNA samples containing 1000 ng faecal DNA are shown
in Fig. 1. The presence
of additional faecal DNA was shown not to affect the sensitivity of the hybridization
probes.
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DISCUSSION |
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Dot-blot hybridization with rRNA-targeted oligonucleotide probes is a well-established
method for studying faecal microbes (Doré et al., 1998; Sghir et al., 2000
; Hopkins et al., 2001
; Marteau et al., 2001
). rRNA is present in abundance
in bacterial cells, thus increasing the sensitivity of the dot-blot hybridization
to a reasonable level. In this study, dot-blot hybridization was performed
with rDNA-targeted oligonucleotide probes to quantify the same starting material
as was used for the real-time PCR applications tested here. The dot-blot hybridization
could, at best, detect a 3 % DNA subpopulation within mixed
DNA samples (Fig. 1
).
A similar sensitivity level has been reported by Muttray & Mohn (2000)
for measurements of rDNA-to-rRNA
ratios, thus the result was as expected, being approximately 10-fold less
sensitive than what is obtained with the rRNA-targeted oligonucleotides (Sghir et al., 2000
).
Real-time PCR applications have been described for the quantification of
total bacteria (Suzuki et al.,
2000; Bach et al., 2002
; Nadkarni et al.,
2002
) and archaea (Suzuki et al., 2000
) present in various environments,
as well as for the sensitive and accurate detection and quantification of
pathogenic bacteria (Nogva et al., 2000
; Hein et al., 2001
; Ge et al.,
2001
). To our knowledge, real-time PCR assays for studying
members of the normal GI tract microflora have not been described. The sensitivity
levels of the real-time PCR assays described in this study were between 200
and 400 target bacteria in pure cultures or mixed DNA samples, depending on
the genome size of the target bacterium (Figs 4 and 5
).
Results obtained by spiking faeces with various amounts (109105 cells g-1) of target bacteria (Fig. 6a,
b
) showed that real-time
PCR is suitable for the analysis of real samples and is highly sensitive.
The SYBR Green I and TaqMan assays used here were both shown to
be sensitive and specific methods for the quantification of target bacteria. Hein et al. (2001) used
a hot-start enzyme to prevent primer dimer formation in SYBR Green I-based
assays, which led to subsequent improvements in the sensitivity of these assays;
however, an even better result was reported for TaqMan-based assays
using the same hot-start polymerase. Here, it was found that the sensitivities
of both chemistries were alike, but a hot-start polymerase was used in the
SYBR Green I assays and a cost-effective standard polymerase was used in the TaqMan assays. Although for the TaqMan-based assays this probably
led to a somewhat less sensitive result than would have been achieved using
a hot-start enzyme, the acquired level of sensitivity, providing a means for
the quantification of a 0·01 % subpopulation in a DNA
sample, was considered sufficient for studying the GI tract ecology. Considering
the inaccuracies that are likely to be introduced into results by the processes
used to isolate DNA from population samples and by the subsequent measurement
of the concentration of template DNA present in a given sample, the observed
accuracy level of real-time PCR in the quantification of target genomes present
in the in vitro DNA mixtures or faecal samples with added target
cells was satisfactory (Figs 4, 5 and 6
).
Genome sizes and rRNA gene copy numbers are also likely to contribute to the
amplification of different target bacteria.
When compared to rDNA-targeted dot-blot hybridization, real-time PCR had a superior sensitivity and also proved to be a more convenient and less expensive method for the quantification of selected bacterial populations. While a reasonable level of sensitivity was obtained here with the TaqMan chemistry using a standard, non-hot-start polymerase, the non-selective detection of any double-stranded DNA molecule by SYBR Green I-based assays can also be considered useful, especially when common primers are used for the quantification of diverse target DNA populations that have no suitable conserved sequence for the design of a detection probe.
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ACKNOWLEDGEMENTS |
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Received 30 August 2002;
accepted 1 October 2002.