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Applied and Environmental Microbiology, November 2000, p. 4605-4614, Vol. 66, No. 11
Monterey Bay Aquarium Research Institute,
Moss Landing, California 95039
Received 15 July 2000/Accepted 14 August 2000
Few techniques are currently available for quantifying specific
prokaryotic taxa in environmental samples. Quantification of specific
genotypes has relied mainly on oligonucleotide hybridization to
extracted rRNA or intact rRNA in whole cells. However, low abundance
and cellular rRNA content limit the application of these techniques in
aquatic environments. In this study, we applied a newly developed
quantitative PCR assay (5'-nuclease assay, also known as
TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs)
from uncultivated planktonic prokaryotes in Monterey Bay. Primer and
probe combinations for quantification of SSU rDNAs at the domain and
group levels were developed and tested for specificity and
quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus
Prochlorococcus and marine Archaea and compared
the results of the quantitative PCR assays to those obtained by
alternative methods. The 5'-nuclease assays reliably quantified rDNAs
over at least 4 orders of magnitude and accurately measured the
proportions of genes in artificial mixtures. The spatial and temporal
distributions of planktonic microbial groups measured by the
5'-nuclease assays were similar to the distributions estimated by
quantitative oligonucleotide probe hybridization, whole-cell
hybridization assays, and flow cytometry.
Ribosomal RNA genes (rDNAs) are
extensively used to study the diversity of microorganisms in
environmental samples. To date, most surveys of microbial diversity in
environmental samples have relied upon cloning and sequencing of rDNAs.
These studies have shown that the diversity of microorganisms in
natural ecosystems has been severely underestimated in culture
collections and have led to the discovery of several new microbial
lineages (5, 10, 20, 28). Despite the increased knowledge of
the phylogenetic diversity of indigenous microbes, less is known about
the abundance of particular groups or their spatial and temporal dynamics.
Few techniques are currently available for quantifying specific
prokaryotic taxa in environmental samples. Recoveries of PCR-amplified rDNAs from clone libraries are subject to several quantitative biases
(see reference 26 for a review). rRNA-based
quantification therefore has relied mainly on radiolabeled
oligonucleotide hybridization to extracted rRNA (11, 16, 21)
or whole-cell hybridization using fluorescence-labeled oligonucleotides
(1, 7). However, in many ecosystems the abundance of
microorganisms is relatively low and many cells may have a low rRNA
content, limiting the sensitivity of oligonucleotide hybridization
techniques for quantification of specific microorganisms.
In individual cells, the number of copies of rDNAs may be several
orders of magnitude lower than that of rRNAs; in DNA, rRNA coding
sequences exist in a context of much higher genetic complexity. It is
therefore difficult to quantify rDNAs by oligonucleotide hybridization.
However, rDNA is suitable for amplification by the PCR. Provided that
the amplification efficiencies of different rDNAs are comparable, the
proportions of specific genes in PCR amplification products should
reflect the proportions of the same rDNAs in the original samples.
Most described PCR-based quantification methods (e.g., dot blot
hybridization [11], denaturing gradient gel
electrophoresis [17], terminal restriction fragment
length [TRFLP] analysis [14], and PCR amplicon
length heterogeneity [LH-PCR] analysis [23]) rely on
quantification after a number of replication cycles have been completed
and reaction products are at concentrations sufficiently high to allow
end-point detection. However, recent studies (23, 24) have
shown that the accumulation of amplification products may bias the
proportions of different amplicons in mixtures compared to the
proportions in the original samples and have suggested that a
minimum number of cycles be used. Furthermore, some of these
techniques (TRFLP and LH-PCR) rely on a diagnostic fragment size,
so microbial identification is presumptive, not determinative.
To circumvent the quantitative difficulties associated with other
methods, we applied a recently developed PCR-based 5'-nuclease assay
(also known as TaqMan [15]) to the quantification of
SSU rDNAs in artificial mixtures and in environmental DNA samples. In
contrast to other PCR-based techniques, this method detects amplified
rDNAs in early cycles of the PCR, and quantification is performed
during the exponential phase of the reactions. The method also allows
the estimation of PCR amplification efficiency.
In this study, we developed general 5'-nuclease assays for the
measurement of rDNAs belonging to the domains Archaea and
Bacteria. We also designed specific assays for the
quantification of SSU rDNAs of bacteria belonging to the genera
Synechococcus and Prochlorococcus and two groups
of uncultivated marine Archaea. We tested the assays using
artificial DNA mixtures from cultivated strains as well as DNA
extracted from bacterioplankton assemblages collected in California
coastal waters. The results indicate that these assays can accurately
estimate relative proportions of genes in complex mixtures. Our results
also revealed some of the inherent limitations of the approach.
Principles of 5'-nuclease assays.
The 5'-nuclease assays are
based on the measurement of the fluorescence intensity of fluorochrome
molecules that are produced during the extension step of PCR by the
5'-to-3' exonuclease activity of Taq DNA polymerase. These
fluorochromes are derived from an oligonucleotide probe with sequence
complementary to a region between the amplification primers (TaqMan
probe). In the intact probe, fluorescence emitted by the 5' reporter
dye is quenched by a dye attached to the 3' end of the probe. During
the PCR amplification cycle, the 5' reporter dye is removed from the
template-bound oligonucleotide by Taq DNA polymerase. The
accumulation of cleaved reporter dye molecules is proportional to the
initial copy numbers of the target genes in template DNA. Target gene
copy numbers in an unknown sample are calculated from the number of
cycles necessary for the fluorescence emission of reporter dyes to
exceed a set threshold value (threshold cycle number
[CT]) relative to standard controls with known numbers of
copies. Slopes of standard curves (regression lines of CT
versus log N, the log of initial gene copy numbers in
standard templates) can be used to estimate PCR amplification
efficiency in the assays. For a detailed description of the method, see
reference 15.
Environmental DNA samples.
Water samples (4 liters) were
collected at five depths at station M1, located 10.8 mi west of Moss
Landing, Calif., on 3 December 1997 and 7 January 1998. Picoplankton
collection and nucleic acid extraction were performed as previously
described (16). DNA was further purified from inhibitory
substances and RNA by CsCl buoyant equilibrium centrifugation with
300-µl tubes and an Optima TL ultracentrifuge (Beckman, Palo Alto,
Calif.) as described previously (3), except that Centricon
100 centrifugal filter units (Millipore, Bedford, Mass.) were used to
remove the CsCl from the DNA sample. All genomic DNA samples were
stored at Cultivated strains.
Several cultivated organisms belonging
to the domains Archaea and Bacteria were chosen
to test primer and probe specificity and to serve as templates for the
optimization of 5'-nuclease assays. Our choice of organisms was based
on phylogenetic relationships and the availability of information on
genome size and number of operons in the genome. All organisms used in
this study are listed in Table 1. Nucleic
acids were extracted from axenic cultures by the cetyltrimethylammonium
bromide (CTAB) protocol (3), and DNA was separated from RNA
by CsCl buoyant equilibrium centrifugation and stored as described
above. Nucleic acids from Synechococcus strains WH7805 and
WH8103 were kindly provided by D. Distel, Department of Biology,
University of Maine. Genomic DNA concentrations were measured
fluorometrically by PicoGreen (Molecular Probes, Eugene, Oreg.)
staining with a FluorImager fluorescence imager (Molecular Dynamics,
Sunnyvale, Calif.) according to the manufacturer's specifications.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Quantitative Analysis of Small-Subunit rRNA Genes
in Mixed Microbial Populations via 5'-Nuclease Assays
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
20 to 80°C in pH 8.0 TE buffer (10 mM Tris-HCl, 1 mM
EDTA) until analysis.
TABLE 1.
Summary of primer specificity tests by three-step
end-point PCR
Plasmid clones. (i) Environmental libraries. Cloned rRNA genes were chosen as controls for all assays. This choice was based on the fact that the number of copies of the gene of interest can be more easily inferred from plasmid concentrations than from genomic DNA concentrations. Also, for many microorganisms, rDNA copy number, genome size, or both have not been determined. Finally, plasmid clones are also the only source of rDNA from uncultivated microorganisms.
Archaeal rDNA standards consisted of a cloned DNA fragment containing the rDNA from a planktonic crenarchaeote (Fosmid 4B7 [22]) and a small-subunit (SSU) rDNA clone from a marine euryarchaeote from the Santa Barbara Channel (SB95-72 [16]). Bacterial rDNA clones were retrieved from a PCR library constructed with a surface seawater DNA sample collected on 5 June 1997 at station S297-67-90 (124°89/W, 35°44/N), 275 km from Moss Landing. Nucleic acids were extracted as previously described (16), and the DNA was purified by CsCl buoyant equilibrium centrifugation and resuspended in 200 µl of TE buffer, pH 8.0. DNA fragments containing nearly the entire SSU rDNA, the intergenic transcribed spacer (ITS), and about 1,900 bp of the SSU rDNA were amplified by PCR. In a final 50-µl volume, reaction mixtures contained 0.5 µl of TaqPlus Long 10× low-salt buffer (Stratagene, La Jolla, Calif.), 0.2 mM each deoxynucleoside triphosphate (dNTP) (Promega, Madison, Wis.), 0.5 µM each primer, 2 mM MgCl2, 4 µl of environmental DNA sample, and 2.5 U of TaqPlus Long polymerase mixture (Stratagene). The forward primer used was the SSU rDNA bacterial primer 27F (5'-AGA GTT TGA TCM TGG CTC AG-3') (9), and the reverse primer used was the large-subunit (LSU) rDNA bacterial primer 1933eR (5'-ACC CGA CAA GGA ATT TCG C-3') (2). A PE9700 thermal cycler (PE Biosystems Inc., Foster City, Calif.) was programmed to 5 min of precycling at 94°C and 30 cycles of 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 3 min. PCR products of triplicate reactions were combined and purified by phenol-chloroform extraction followed by ethanol precipitation (19), resuspended in 20 µl of deionized water, and blunt ended by treatment with Pfu DNA polymerase. In a final volume of 10 µl, the blunt-ending reaction mixtures contained 1 µl of recombinant Pfu DNA polymerase 10× buffer (Stratagene), 1 mM each dNTP, and 0.5 U of recombinant Pfu DNA polymerase (Stratagene). Reaction mixtures were incubated at 94°C for 20 min in a PE9700 thermal cycler. Products of the blunt-ending reaction were purified by phenol-chloroform extraction followed by ethanol precipitation and resuspended in 10 µl of deionized water. Two microliters of the purified blunt-ended PCR product was ligated into the vector PCR Zero Blunt (Invitrogen, Carlsbad, Calif.) using the manufacturer's protocol, and the resulting ligation product was used to transform Escherichia coli One Shot TOP10 competent cells (Invitrogen). A total of 384 white colonies were picked, and plasmids were archived in 96-well microtiter dishes as previously described (3). A total of 190 clones were screened and grouped based on HaeIII restriction fragment length polymorphism analysis as previously described (25). Clones representing all HaeIII restriction fragment length polymorphism patterns were bidirectionally sequenced by the dideoxy termination reaction using an LC7200 automated DNA sequencer (LI-COR, Lincoln, Nebr.). These clones have the prefix MB1. Archaeal clones from additional libraries (5, 16) were used for the primer specificity tests (Table 1). These plasmids were purified using a Qiaprep Spin plasmid kit (Qiagen, Valencia, Calif.) or a Mini-Prep 24 machine (MacConnell Research, La Jolla, Calif.). Plasmids used as standards for the 5'-nuclease assays were purified using a Qiagen Maxi plasmid kit according to the manufacturer's protocol and further purified by CsCl buoyant equilibrium centrifugation with 3.6-ml tubes and an Optima TL ultracentrifuge as described previously (3), except that Centricon 100 centrifugal filter units were used to remove the CsCl. Linearized plasmids were produced by digestion with the restriction endonuclease NotI (Promega) according to the manufacturer's protocol, purified by phenol-chloroform extraction, and subjected to three washes using Microcon 100 centrifugal filter units (Millipore) and a final desalting step using Micro Bio-spin columns (Bio-Rad, Hercules, Calif.) loaded with TE buffer. DNA concentrations of purified linearized plasmids were determined fluorometrically as described above. Supercoiled (not linearized) plasmid concentrations were determined spectrophotometrically.(ii) Cultivated organisms.
The plasmid containing the
SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the
-Proteobacteria (25) (R57pCRbl), was prepared
from extracted genomic DNA in the same manner as the environmental
clones, except that the DNA polymerase used for amplification was
TaqPlus Precision DNA polymerase mix (Stratagene) and the
PCR products were directly cloned using a PCR ZeroBlunt kit
(Invitrogen). The plasmid containing the SSU-ITS-LSU rDNA fragment of
Haloferax volcanii (HvpCRbl) was produced in a similar fashion, except that the primers used were the degenerate archaeal SSU
rDNA forward primer 21F (5'-TTC CGG TGG ATC CYG CCG GA-3' [16]) and the degenerate prokaryotic LSU rDNA reverse
primer 2445R (5'-CCC YGG GGT ARC TTT TCT ST-3' [6]).
These plasmids were purified using a Qiagen Maxi plasmid kit and
isolated from genomic DNA via CsCl buoyant equilibrium centrifugation
(3).
Primers and probes.
The TaqMan probe and reverse primers for
the Bacteria and Archaea assays targeted the
previously described universal regions homologous to Escherichia
coli positions 1392 to 1406 (13) 1492 to 1510 (13), and 1518 to 1541 (9) (Table
2). Our PCR primers and probes were
designed using ARB software graciously provided by O. Strunk and W. Ludwig, Technical University of Munich, Munich, Germany. A database of
over 10,000 SSU rRNA sequences was used to check primer specificity and
possible mismatches. Although all universal primers showed significant
mismatches with some major bacterial and archaeal groups (Table 2), we
opted to exclude the detection of genes belonging to these groups
rather than to increase the numbers of degenerate positions in the
primers, which would complicate the measurement of effective primer
concentrations in the reactions. Group-specific primers and probes were
designed using ARB software (Table 2). Since the target organisms were closely related, in the majority of cases it was possible to design primers with no degeneracies. All probes and primers were screened and
optimized for the requirements of 5'-nuclease assays using Primer
Express software (PE Biosystems).
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Performance of 5'-nuclease assays.
Although optimized
5'-nuclease assay parameters (i.e., primers, fluorogenic probe, and
MgCl2 concentrations) between different primer and probe
sets varied, the following conditions were identical for all. In a
final volume of 25 µl, reaction mixtures contained the buffer
recommended for the DNA polymerase used, 200 µM each dATP, dCTP, and
dGTP, 400 µM dUTP, and 0.25 U of AmpErase uracyl N-Glycosylase (UNG; PE Biosystems). For all of the primer
and probe sets, except for BACT2, 0.025 U of AmpliTaq Gold
DNA polymerase (PE Biosystems) µl
1 was used. For the
BACT2 set, 0.025 U of Platinum Taq DNA polymerase (Life
Technologies, Rockville, Md.) µl1 was used. All
reactions were carried out with optical tubes or reaction trays (PE
Biosystems), with 2.5 µl of a template being delivered into the tubes
first using a Microman M10 positive-displacement pipette (Rainin,
Emeryville, Calif.). A master mix (22.5 µl) was delivered using a
Microman M100 positive-displacement pipette (Rainin), and tubes were
sealed with optical caps. In experiments optimizing the effect of
primer, probe, or MgCl2 concentrations, these reagents were
excluded from the master mix and added to the tubes last. All reactions
were performed with a model 7700 sequence detection system (PE
Biosystems) programmed with a soak step of 2 min at 50°C, allowing
AmpErase UNG to hydrolyze PCR amplicons possibly carried over from
previous reactions. An enzyme activation soak step (15 min at 95°C
for AmpliTaq Gold or 2 min at 94°C for Platinum
Taq) followed the initial soak step. Finally, 40 cycles of
15 s of denaturation (95°C for AmpliTaq Gold or
94°C for Platinum Taq) and 1 min of annealing plus
extension at the temperatures listed in Table 2 were performed. All
results were analyzed with a PowerMac 4400 (Apple Computer Co.,
Cupertino, Calif.) computer using Sequence Detector v1.6.3 software (PE Biosystems).
Optimization of 5'-nuclease assays. (i) Primer melting
temperature.
Since the BACT1 and ARCH1 primer and probe sets
shared the same reverse primer and 5'-nuclease probe, we empirically
tested the melting temperature of all of our primers via end-point PCR. In a final volume of 20 µl, reaction mixtures contained 1 µl of AmpliTaq Gold 10× buffer (PE Biosystems), 200 µM each
dATP, dCTP, and dGTP, 400 µM dUTP, 1.5 mM MgCl2, 0.2 ng
of template DNA, 0.05 U of AmpErase UNG, and 0.025 U of
AmpliTaq Gold DNA polymerase µl1. We used
E. coli DH10B DNA to test the bacterial primer set and Methanobacterium thermoautotrophicum and Thermoplasma
acidophilum to test the archaeal primer set. Reactions were run in
a Robocycler Gradient96 thermal cycler (Stratagene) programmed to a
10-min enzyme activation soak step at 94°C and 25 cycles of 95°C
denaturation for 52 s, a gradient of 56 to 65°C annealing for
the bacterial primer set or 53 to 65°C annealing for the archaeal set
for 30 s, and 72°C extension for 42 s. Mineral oil was
overlaid on the samples to avoid evaporation, and 10 µl of the
reaction products was run in 1% agarose minigels stained with ethidium
bromide (50 µg/ml). Gels were scanned with an MD FSI fluorescence
imager (Molecular Dynamics). We tested the melting temperature of the
BACT2 set in a similar manner, except that the final reaction volume
was 25 µl, the template used was strain R2A57 genomic DNA, and the gradient was 52 to 63°C.
(ii) Specificity. Since most TaqMan probes were designed to target broad groups of organisms (i.e., prokaryotes or marine Archaea), we relied on the sequences of the primers for specificity. To examine possible cross-reactivity of the primers, we initially examined the formation of PCR products after 25 cycles in three-step PCRs. In a final volume of 10 µl, reaction mixtures contained 1 µl of AmpliTaq Gold 10× buffer, 0.2 mM each dNTP, 0.5 µM each primer, 1.5 mM MgCl2, and 0.05 U of AmpliTaq Gold DNA polymerase. Reactions were run in a PE9700 thermal cycler at the annealing temperatures listed in Table 1.
(iii) Primer concentration. 5'-Nuclease assay reactions were performed using a matrix of concentrations of forward and reverse primers to seek primer concentrations yielding minimal CT values and consequently the highest amplification efficiencies. Primer concentrations ranged from 100 to 1,500 nM. Annealing temperatures as well as MgCl2 concentrations were the same as those listed in Table 2. The remaining parameters were identical to those described above. For primer and probe sets using universal primers (i.e., PROK1541R and PROK1492R) or probes (i.e., ARCH519R and PROK1389F), we used specific target genomic or plasmid DNA alone as well as mixtures of target and nontarget genomic DNAs.
(iv) Probe concentration. 5'-Nuclease assay reactions were performed using optimized primer concentrations and a TaqMan probe concentration range. We determined the probe concentration yielding the minimal CT value for a specific template. Probe concentrations ranged from 200 to 800 nM, and the remaining reaction conditions were the same as used for the primer matrices. We used pure target genomic DNA as well as mixtures of target and nontarget genomic DNAs for primer and probe sets using universal probes (i.e., ARCH519R and PROK1389F), since the probes are complementary to both target and nontarget DNAs.
(v) MgCl2 concentration. MgCl2 concentration has two main effects on PCRs. First, MgCl2 is required for Taq DNA polymerase activity. This was particularly important in 5'-nuclease assays, since the extension step was performed at suboptimal temperatures for the DNA polymerase. On the other hand, MgCl2 concentration affects the melting temperature of the primers and consequently their specificity. This was particularly critical for the BACT1, BACT2, and ARCH1 primer and probe sets, which relied only on the forward primers for specificity. For primer and probe sets BACT1 and ARCH1, we chose the minimal MgCl2 concentrations that allowed consistent amplification of templates at different concentrations and reasonably high amplification efficiencies, estimated from the slopes of standard curves empirically generated by 5'-nuclease assays. We used the same MgCl2 concentrations for primer and probe sets BACT2 and ARCH2, and 5 µM MgCl2 (final) was used for the remaining 5'-nuclease assay primer and probe sets.
Effect of template source and type.
To test whether
genomic DNAs from different organisms might be differentially
amplified by 5'-nuclease assays, standard curves for E. coli, Bacillus subtilis, and Deinococcus
radiodurans genomic DNAs were compared. The 25-µl
reaction mixtures contained 2.5 µl of AmpliTaq Gold 10×
buffer, 1.5 mM MgCl2, 200 µM each dATP, dCTP, and dGTP,
400 µM dUTP, 500 nM each primers BACT1369F and PROK1541R, 200 nM
probe TM1389F, 0.05 U of AmpErase UNG, 2.5 µl of templates with copy
numbers ranging from 104 to 107 copies
µl1, and 0.025 U of AmpliTaq Gold DNA
polymerase µl1. Reactions were performed with a model
7700 sequence detection system and the cycling parameters were the same
as those already described. Triplicate standard curves for each of the
genomic DNA types were compared by analysis of variance (ANOVA).
Mixed-template experiments.
To test the applicability of
5'-nuclease assays for relative quantification, artificial mixtures of
SSU rDNAs were prepared. In the first test, we used mixtures of strain
R2A57 and H. volcanii genomic DNAs. R2A57 SSU rDNA
copy numbers were estimated using the BACT1 set, and H. volcanii SSU rDNA copy numbers were estimated using the ARCH1 set.
Plasmids R57pCRbl and HVpCRbl were used as standards. Reactions were
carried out with the optimized conditions listed in Table 2. Three
experiments were conducted with genomic DNA mixtures
(106 SSU rDNA copies µl1) adjusted to the
following proportions of H. volcanii to R2A57: 0.1:0.9,
0.2:0.8, 0.4:0.6, 0.6:0.4, 0.8:0.2, and 0.9:0.1. In the second test, we
used mixtures of strain R2A57 and Synechococcus sp. strain
WH7803 genomic DNAs. Copy numbers of Synechococcus SSU rDNA were estimated using the PHPICO primer and probe set, the
plasmid clone MB11A04 as a standard, and the optimized conditions listed in Table 2. Total bacterial SSU rDNAs were measured with both
BACT1 and BACT2 primer sets using R57pCRbl and MB11A04 as standards.
The same proportions of SSU rDNA and calculations were used.
Validation of 5'-nuclease assays for analyzing environmental samples. To validate the use of 5'-nuclease assays as a quantitative tool for complex environmental samples, we compared results obtained with alternative methods. First, we estimated the proportion of SSU rDNA belonging to the Synechococcus and Prochlorococcus group relative to bacterial SSU rDNAs in Monterey Bay DNA extracts using the 5'-nuclease assays versus flow cytometry cell counts. Flow cytometry cell counts were determined at the Department of Oceanography, University of Hawaii, as previously described (4).
In a second set of experiments, the relative abundance of SSU rDNAs from group I and II marine Archaea in Monterey Bay DNA extracts was estimated by 5'-nuclease assays and compared to the results obtained using fluorescent in situ hybridization (FISH [6]) or RNA blot hybridization (16) for the same samples. SSU rDNA copies of group I marine Archaea in environmental samples from the Monterey Bay were estimated using the ARCHGI 5'-nuclease set and the optimized conditions listed in Table 2. SSU rDNA copies of group II marine Archaea were estimated using the ARCHGII primer set and clone SB9572 as a standard. FISH experiments and RNA blot hybridization experiments were performed as previously described (6, 16).Analytical considerations. The analytical precision of the model 7700 SDS instrument was relatively high. In general, triplicate measurements obtained simultaneously had a coefficient of variation of less than 0.15 (average, 0.092; n = 52) for all primer and probe sets tested (BACT1, BACT2, ARCH1, and PHPICO), although on rare occasions it was as high as 0.42. However, day-to-day variability in copy numbers estimated with the primer and probe sets BACT1, ARCH1, PHPICO, ARCHGI, and ARCHGII was much higher for replicate samples. In most cases, the coefficients of variation were higher than 0.30 (average, 0.74; n = 30).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Laboratory optimization of 5'-nuclease assays. Initially we designed two broad primer and probe sets targeting Archaea and Bacteria (BACT1 and ARCH1). As discussed below, these primer and probe sets were shown to underestimate a number of groups that are present in a relatively high abundance in environmental samples; therefore, their utility for these samples appears limited. However, these primer and probe sets were very useful for optimizing and testing critical parameters of the 5'-nuclease assays.
(i) Specificity of and optimal conditions for 5'-nuclease assays. The primer and probe sets BACT1 and ARCH1 utilized a common probe (PROK1389F) and a reverse primer (PROK1541R). This strategy was used to help standardize assays and to avoid the synthesis of multiple TaqMan probes. We relied on the specificity of forward primers to discriminate between rDNAs of bacterial and archaeal origins. Specificity tests showed that both primer and probe sets were specific for their intended groups. Optimal reaction conditions for all primer and probe sets are listed in Table 2.
(ii) Effect of template source and type.
There were no
significant differences in the slopes of standard curves for
genomic DNAs from E. coli, B. subtilis,
or D. radiodurans, as determined by ANOVA (Table
3; = 0.05). The
t-test comparison (29) between the slopes of
standard curves obtained with supercoiled plasmids, linear plasmids,
and genomic DNA containing SSU rDNA from strain R2A57 or
H. volcanii also showed no significant differences ( = 0.05). Since gene copy numbers were more accurately calculated from
plasmid DNA concentrations, we also compared the intercepts of standard
curves for linear and supercoiled R57pCRbl and HvpCRbl. Comparison by
t tests (29) revealed significant differences ( = 0.05) between the intercepts of supercoiled and linear
HvpCRbl (Fig. 1) and R57pCRbl (data not
shown) standard curves and underestimation of the copy numbers of
supercoiled plasmids relative to linear plasmids by about 1 order of
magnitude.
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(iii) Detection limits. BACT1 consistently produced a high background fluorescence signal in negative controls containing no template. This background signal was equivalent to the signal produced by approximately 2,500 SSU rDNA copies. Reactions carried out with both AmpliTaq Gold and Platinum Taq had equivalent backgrounds, and it is likely that the background originated from host DNA carryover in the cloned DNA polymerase preparations. Attempts to decrease the background by incubating the cocktail mixtures (without a template and primers) with exonucleases failed. Therefore, we assigned 25,000 copies of template per reaction tube as our detection limit for the bacterial domain primer and probe sets. The range for bacterial quantification was 2.5 × 104 to 2.5 × 108 copies per reaction tube, although higher values might still lie in the regression line of standard curves. In the majority of cases, the archaeal set ARCH1 produce no fluorescence signal in negative controls containing no template. The quantification range for archaeal and other group-specific assays was 2.5 × 102 to 2.5 × 108 copies (0.14 to 1.4 × 104 pg) per reaction tube, based on the genome size and rrn operon copy numbers in E. coli.
(iv) Mixed-template experiments.
Primer and probe sets BACT1
and ARCH1 were successfully used to test the accuracy of estimating SSU
rDNA percentages in genomic DNA mixtures. The percentage of
H. volcanii genomic DNA in artificial mixtures with
strain R2A57 genomic DNA estimated by the 5'-nuclease assays
was not significantly different from the proportion in the templates
(Fig. 2). We also used BACT1 to estimate
total SSU rDNA copy numbers in artificial mixtures of genomic
DNAs from Synechococcus strain WH7803 and strain R2A57.
Surprisingly, the proportion of Synechococcus strain WH7803
estimated by the PHPICO set was overestimated twofold in mixtures when
the percentage of Synechococcus strain WH7803 DNA in the
template was high (over 80%). This result was clearly caused by an
underestimation of total bacterial SSU rDNA by BACT1, likely due to
mismatches with PROK1541R (3a; M. T. Suzuki et
al., unpublished data). There are a limited number of public database
sequences that include this SSU rDNA region, and some of the existing
sequences are those of the PCR primers used to recover the SSU rDNA.
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Field application of 5'-nuclease assays. We designed four additional primer and probe sets (BACT2, PHPICO, ARCHGI, and ARCHGII) to test the applicability of the 5'-nuclease assays to estimation of the proportions of different groups of prokaryotes in environmental DNA samples. Based on our initial tests and observations, we used the following procedure when applying 5'-nuclease assays to the quantification of prokaryotic rDNAs. (i) Quantification was performed relative to DNA mass or to rDNA copy numbers estimated by a domain-level primer and probe set. (ii) Linear plasmids were used as standards. (iii) Whenever possible, the same standards were used for group-specific and domain-specific primer and probe sets.
(i) Bacteria.
Since primer PROK1541R appears to miss several
major groups of Bacteria, we used primer PROK1492R as the
reverse primer for the bacterial assay for environmental samples. This
primer has a melting temperature (56°C), which is lower than the
optimal temperature for Taq DNA polymerase (72°C), more so
than BACT1 (59°C). Also, we observed considerable variability in the
activity of AmpliTaq Gold DNA polymerase, possibly caused by
variability in enzyme activation (data not shown). Therefore, we used
Platinum Taq DNA polymerase in assays with primer PROK1492R.
The BACT2 set was tested for estimating the proportions of
Synechococcus strain WH7803 genomic DNA in mixtures
with strain R2A57. Results showed good agreement between the expected
and observed proportions of Synechococcus strain WH7803
(Fig. 3). The BACT2 set was also used to
estimate SSU rDNAs from Bacteria in samples from Monterey Bay. In this case, the proportions of SSU rDNAs of
Synechococcus and Prochlorococcus estimated by
the PHPICO set were similar to those estimated by cell counts, with the
exception of surface and 20-m samples (Fig.
4). It is possible that this discrepancy between the proportions estimated by cell counts and 5'-nuclease assays
was due to an underestimation of photoadapted cells by flow cytometry,
which has been previously reported (18). As with BACT1, the
range of quantification was 2.5 × 104 to 2.5 × 108 copies per reaction tube.
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(ii) Synechococcus and Prochlorococcus. Set PHPICO was designed to target SSU rDNAs of Synechococcus and Prochlorococcus and to amplify a region homologous to positions 191 to 420 of E. coli SSU rDNA (Table 1). We used this probe to measure SSU rDNA of Synechococcus strain WH7803 in artificial mixtures with strain R2A57 and to estimate the proportions of Synechococcus and Prochlorococcus genes in DNA samples from Monterey Bay. The estimated proportions of SSU rDNAs correlated well with expected proportions. The tested range of quantification of the PHPICO primer and probe set was 2.5 × 103 to 2.5 × 107 SSU rDNA copies per reaction tube.
(iii) Marine Archaea. Although we tested several different combinations of primer and probe sets targeting archaeal SSU rDNAs, currently we have no broadly encompassing set that is suitable for application to all environmental samples. Forward primer Arch1369F has several mismatches with SSU rDNAs of Methanosarcinales and Methanococcales, so that these groups likely are underestimated by the set containing this primer. Since these organisms were not likely to represent a large fraction of the archaeal population in planktonic samples, we used ARCH1 to estimate total archaeal SSU rDNAs in samples from Monterey Bay. The proportions of SSU rDNA of group I marine Archaea estimated by ARCHGI relative to total archaeal SSU rDNAs were similar to those estimated by rRNA blot hybridization or FISH. However, the proportions of SSU rDNA of group II marine Archaea estimated by ARCHGII were overestimated by 2 orders of magnitude, likely due to mismatches between group II marine Archaea SSU rDNA and the reverse primer PROK1541R (data not shown). Currently, there is no sequence information for the 3' end of the SSU rDNA of group II marine Archaea, since the only sequences available are those from PCR clones with no sequence for this region of the SSU rDNA.
Although we encountered difficulties designing a primer and probe set suitable for all Archaea, we designed two primer and probe sets to target SSU rDNAs of two abundant groups of marine Archaea (Table 1). Set ARCHGI targeted genes of the marine Crenarchaeota (group I), and set ARCHGII targeted genes of the marine Euryarchaeota (group II). The ratios of SSU rDNAs from group I and II Archaea correlated well with the ratios of their rRNAs estimated in dot blot experiments using radiolabeled oligonucleotide probes (Fig. 5). These ratios also correlated well with those estimated from cell counts by polyribonucleotide FISH and agree with previous observations of the depth distribution of these groups (16). The range of quantification of both sets was 2.5 × 103 to 2.5 × 107 gene copies per reaction tube.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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We successfully developed and tested PCR-based fluorogenic 5'-nuclease assays for the relative quantification of SSU rDNAs of prokaryotes. It was possible to attain high sensitivity using small amounts of a sample, and the quantification range exceeded 4 orders of magnitude. These characteristics of 5'-nuclease assays have important implications for sampling strategies and experimental design in microbial ecology. The sensitivity and high throughput of the assays allow genes in DNA from small biomass samples to be detected and a large number of samples to be processed. Although we encountered difficulties in designing primer and probe sets that encompass entire domains, it was relatively easy to design primer and probe sets for rDNAs for narrower taxonomic groups. In the absence of domain-specific primer and probe sets for normalization, taxon-specific genes can be quantified relative to total DNA.
We attempted only relative quantification, for several reasons. (i) The quantification of SSU rDNA copy numbers in genomic DNA from DNA concentration requires a priori knowledge of rDNA copy number and genome size. Also, in the few instances when both values are known, a cell might contain more than one genome copy and consequently extra copies of the rrn operon. Therefore, gene copy numbers in stock solutions of genomic DNA were calibrated relative to gene copy numbers in plasmid DNA solutions using 5'-nuclease assays. (ii) We used linear plasmids as standards. Copy numbers were estimated from DNA concentrations in samples quantified by fluorometry. We did not attempt to improve the accuracy of copy number measurements by alternative methods (e.g., Poisson distribution [27]), since a further increase in the accuracy of copy number measurements was not justified when the next item was taken into consideration. (iii) Copy numbers of SSU rDNA were measured relative to the total amount of DNA extracted and purified from a mixed population. Due to methodological limitations associated with the study of uncultivated microorganisms, the accurate estimation of DNA extraction efficiency and, consequently, the amount of total DNA present in the original seawater sample was not possible. In view of these problems associated with absolute quantification, our strategy was to measure the proportions of rDNAs from different groups of Bacteria relative to all rDNAs detected from the domain Bacteria.
The amplification efficiency was independent of the template type (chromosomal versus plasmid clones); this is a critical consideration in the application of 5'-nuclease assays to environmental samples, since many naturally occurring but uncultured microbial species have not been cultivated, and for these organisms, only cloned rDNAs are available. Additionally, rDNAs from different bacterial phyla were amplified with similar efficiencies, further supporting the applicability of 5'-nuclease assays for mixed populations of bacteria. The significant difference between the y intercepts of standard curves for linear and supercoiled plasmids was likely due to differences in their concentration estimates and underscores the importance of normalization in relative quantification.
Variations in rrn operon copy numbers and genome sizes
between different organisms are complex variables to be considered in
the interpretation of relative proportions of SSU rDNAs in mixed
populations. A recent report (8) reviewed the current information on prokaryotic genome sizes and operon copy numbers and
concluded that genome size and operon copy number are relatively constant in certain phylogenetic groups. The authors suggested that
estimation of organism numbers from SSU rDNA proportions
based on
group averages of genome size and operon copy numbermight be
possible. However, no information is available on genome sizes and
rrn operon copy numbers for many indigenous microorganisms, and in several instances, no cultivated species related to these organisms are available to allow inference of genome sizes and rrn operon copy numbers. Additionally, a recent report by
Klappenbach et al. (12) suggested that rrn operon
copy number may be related to growth strategy and not necessarily to
phylogenetic affiliation. The approach suggested by Fogel et al.
(8) therefore does not seem particularly applicable to
complex, natural microbial assemblages.
By modifying the assays we describe here, it should also be possible to monitor proxies for metabolic activity. For instance, quantification of rRNA after an initial reverse transcription step and comparison of rRNA with rDNA levels could be performed using the same primer and probe sets. Furthermore, it is feasible to measure the relative expression of metabolic genes using reverse transcription of mRNA and PCR quantification by 5'-nuclease assays. Gene expression could then be quantified relative to specific phylogenetic markers (rDNAs or rRNAs), as described in this study. These approaches should provide much better estimates of the metabolic status of microorganisms in situ. Because of their high sensitivity and wide dynamic range, 5'-nuclease assays are particularly suited for samples containing a low level of biomass or slowly growing organisms.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by a grant from the David and Lucile Packard Foundation to MBARI.
We thank D. Distel for providing genomic DNA from Synechococcus strains WH8108 and WH7805 and B. Stevenson for providing clone pKK3535 used in preliminary experiments. We thank the crew of the RV Point Lobos and Tim Pennington for help during sampling. We thank Oded Béjà for editorial comments on the manuscript and Gianfranco de Feo for help during the early stages of development of our 5'-nuclease assays. We also thank two anonymous reviewers for their useful comments.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Monterey Bay Aquarium Research Institute, 7700 Sandholdt Rd., P.O. Box 628, Moss Landing, CA 95039. Phone: (831) 775-1843. Fax: (831) 775-1645. E-mail: delong{at}mbari.org.
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REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Amann, R. I.,
L. Krumholz, and D. A. Stahl.
1990.
Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology.
J. Bacteriol.
172:762-770 |
| 2. |
Amann, R. I.,
W. Ludwig, and K. H. Schleifer.
1995.
Phylogenetic identification and in situ detection of individual microbial cells without cultivation.
Microbiol. Rev.
59:143-169 |
| 3. | Ausubel, F. A., R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman, and K. Struhl. 1988. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y. |
| 3a. | Béjà, O., M. T. Suzuki, E. V. Koonin, L. Aravind, A. Hadd, L. P. Nguyen, R. Villacorte, M. Amjadi, C. Garrigues, S. B. Jovanovich, R. A. Feldman, and E. F. DeLong. Environ. Microbiol., in press. |
| 4. | Buck, K. R., F. P. Chavez, and L. Campbell. 1996. Basin-wide distributions of living carbon components and the inverted trophic pyramid of the central gyre of the North Atlantic Ocean, summer 1993. Aquat. Microb. Ecol. 10:283-298. |
| 5. |
DeLong, E. F.
1992.
Archaea in coastal marine environments.
Proc. Natl. Acad. Sci. USA
89:5685-5689 |
| 6. |
DeLong, E. F.,
L. T. Taylor,
T. L. Marsh, and C. M. Preston.
1999.
Visualization and enumeration of marine planktonic Archaea and Bacteria by using polyribonucleotide probes and fluorescent in situ hybridization.
Appl. Environ. Microbiol.
65:5554-5563 |
| 7. |
DeLong, E. F.,
G. S. Wickham, and N. R. Pace.
1989.
Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.
Science
243:1360-1363 |
| 8. | Fogel, G. B., C. R. Collins, J. Li, and C. F. Brunk. 1999. Prokaryotic genome size and SSU rDNA copy number: estimation of microbial relative abundance from a mixed population. Microb. Ecol. 38:93-113[CrossRef][Medline]. |
| 9. | Giovannoni, S. J. 1991. The polymerase chain reaction, p. 177-203. In E. Stackebrandt, and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Inc., New York, N.Y. |
| 10. | Giovannoni, S. J., T. B. Britschgi, C. L. Moyer, and K. G. Field. 1990. Genetic diversity in Sargasso Sea bacterioplankton. Nature 345:60-63[CrossRef][Medline]. |
| 11. |
Giovannoni, S. J.,
M. S. Rappe,
K. L. Vergin, and N. L. Adair.
1996.
16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the green non-sulfur bacteria.
Proc. Natl. Acad. Sci. USA
93:7979-7984 |
| 12. |
Klappenbach, J. A.,
J. M. Dunbar, and T. M. Schmidt.
2000.
rRNA operon copy number reflects ecological strategies of bacteria.
Appl. Environ. Microbiol.
66:1328-1333 |
| 13. | Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt, and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Inc., New York, N.Y. |
| 14. | Liu, W. T., T. L. Marsh, H. Cheng, and L. J. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63:4516-4522[Abstract]. |
| 15. | Livak, K. J., S. J. Flood, J. Marmaro, W. Giusti, and K. Deetz. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4:357-362[Medline]. |
| 16. | Massana, R., A. E. Murray, C. M. Preston, and E. F. DeLong. 1997. Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel. Appl. Environ. Microbiol. 63:50-56[Abstract]. |
| 16b. | Mullins, T. D., T. B. Britschgi, R. L. Kvest, and S. J. Giovannoni. 1995. Genetic comparisons reveal the same unknown bacterial lineages in Atlantic and Pacific bacterioplankton communities. Limnol. Oceanogr. 40:145-158. |
| 17. |
Muyzer, G.,
E. C. de Waal, and A. G. Uitterlinden.
1993.
Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.
Appl. Environ. Microbiol.
59:695-700 |
| 18. |
Partensky, F.,
W. R. Hess, and D. Vaulot.
1999.
Prochlorococcus, a marine photosynthetic prokaryote of global significance.
Microbiol. Mol. Biol. Rev.
63:106-127 |
| 19. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 20. |
Schmidt, T. M.,
E. F. DeLong, and N. R. Pace.
1991.
Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.
J. Bacteriol.
173:4371-4378 |
| 21. |
Stahl, D. A.,
B. Flesher,
H. R. Mansfield, and L. Montgomery.
1988.
Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology.
Appl. Environ. Microbiol.
54:1079-1084 |
| 22. |
Stein, J. L.,
T. L. Marsh,
K. Y. Wu,
H. Shizuya, and E. F. DeLong.
1996.
Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.
J. Bacteriol.
178:591-599 |
| 23. |
Suzuki, M.,
M. S. Rappe, and S. J. Giovannoni.
1998.
Kinetic bias in estimates of coastal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon length heterogeneity.
Appl. Environ. Microbiol.
64:4522-4529 |
| 24. | Suzuki, M. T., and S. J. Giovannoni. 1996. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62:625-630[Abstract]. |
| 25. | Suzuki, M. T., M. S. Rappé, Z. W. Haimberger, H. Winfield, N. Adair, J. Ströbel, and S. J. Giovannoni. 1997. Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample. Appl. Environ. Microbiol. 63:983-989[Abstract]. |
| 26. | von Wintzingoerode, F., U. B. Gobel, and E. Stackebrandt. 1997. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21:213-229[CrossRef][Medline]. |
| 27. | Wang, Z., and J. Spadoro. 1998. Determination of target copy number of quantitative standards used in PCR based diagnostic assays, p. 31-43. In F. Ferre (ed.), Gene quantification. Birkhauser, Boston, Mass. |
| 28. | Ward, D. M., R. Weller, and M. M. Bateson. 1990. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345:63-65[CrossRef][Medline]. |
| 29. | Zar, J. 1998. Biostatistical analysis, 4th ed. Prentice-Hall, Inc., Upper Saddle Ridge, N.J. |
Applied and Environmental Microbiology, November 2000, p. 4605-4614, Vol. 66, No. 11
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[Abstract] [Full Text] -
Brinig, M. M., Lepp, P. W., Ouverney, C. C., Armitage, G. C., Relman, D. A.
(2003). Prevalence of Bacteria of Division TM7 in Human Subgingival Plaque and Their Association with Disease. Appl. Environ. Microbiol.
69: 1687-1694
[Abstract] [Full Text] -
Brinkman, N. E., Haugland, R. A., Wymer, L. J., Byappanahalli, M., Whitman, R. L., Vesper, S. J.
(2003). Evaluation of a Rapid, Quantitative Real-Time PCR Method for Enumeration of Pathogenic Candida Cells in Water. Appl. Environ. Microbiol.
69: 1775-1782
[Abstract] [Full Text] -
Lueders, T., Friedrich, M. W.
(2003). Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts. Appl. Environ. Microbiol.
69: 320-326
[Abstract] [Full Text] -
Malinen, E., Kassinen, A., Rinttila, T., Palva, A.
(2003). Comparison of real-time PCR with SYBR Green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology
149: 269-277
[Abstract] [Full Text] -
Wawrik, B., Paul, J. H., Tabita, F. R.
(2002). Real-Time PCR Quantification of rbcL (Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase) mRNA in Diatoms and Pelagophytes. Appl. Environ. Microbiol.
68: 3771-3779
[Abstract] [Full Text] -
Pernthaler, A., Pernthaler, J., Amann, R.
(2002). Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria. Appl. Environ. Microbiol.
68: 3094-3101
[Abstract] [Full Text] -
Weinbauer, M. G., Fritz, I., Wenderoth, D. F., Hofle, M. G.
(2002). Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses. Appl. Environ. Microbiol.
68: 1082-1087
[Abstract] [Full Text] -
Rocap, G., Distel, D. L., Waterbury, J. B., Chisholm, S. W.
(2002). Resolution of Prochlorococcus and Synechococcus Ecotypes by Using 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences. Appl. Environ. Microbiol.
68: 1180-1191
[Abstract] [Full Text] -
Nadkarni, M. A., Martin, F. E., Jacques, N. A., Hunter, N.
(2002). Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology
148: 257-266
[Abstract] [Full Text] -
Eguchi, M., Ostrowski, M., Fegatella, F., Bowman, J., Nichols, D., Nishino, T., Cavicchioli, R.
(2001). Sphingomonas alaskensis Strain AFO1, an Abundant Oligotrophic Ultramicrobacterium from the North Pacific. Appl. Environ. Microbiol.
67: 4945-4954
[Abstract] [Full Text] -
Eilers, H., Pernthaler, J., Peplies, J., Glockner, F. O., Gerdts, G., Amann, R.
(2001). Isolation of Novel Pelagic Bacteria from the German Bight and Their Seasonal Contributions to Surface Picoplankton. Appl. Environ. Microbiol.
67: 5134-5142
[Abstract] [Full Text] -
Hristova, K. R., Lutenegger, C. M., Scow, K. M.
(2001). Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR. Appl. Environ. Microbiol.
67: 5154-5160
[Abstract] [Full Text] -
Lim, E. L., Tomita, A. V., Thilly, W. G., Polz, M. F.
(2001). Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells. Appl. Environ. Microbiol.
67: 3897-3903
[Abstract] [Full Text] -
Stults, J. R., Snoeyenbos-West, O., Methe, B., Lovley, D. R., Chandler, D. P.
(2001). Application of the 5' Fluorogenic Exonuclease Assay (TaqMan) for Quantitative Ribosomal DNA and rRNA Analysis in Sediments. Appl. Environ. Microbiol.
67: 2781-2789
[Abstract] [Full Text] -
Grüntzig, V., Nold, S. C., Zhou, J., Tiedje, J. M.
(2001). Pseudomonas stutzeri Nitrite Reductase Gene Abundance in Environmental Samples Measured by Real-Time PCR. Appl. Environ. Microbiol.
67: 760-768
[Abstract] [Full Text]
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