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. 2000 Nov;66(11):4605-14.
doi: 10.1128/AEM.66.11.4605-4614.2000.

Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5'-nuclease assays

M T Suzuki  1 , L T Taylor, E F DeLong
Affiliations

Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5'-nuclease assays

M T Suzuki et al. Appl Environ Microbiol. 2000 Nov.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Effect of DNA quality on the amplification of SSU rDNA by PCR. Standard curves show plots of CT versus log number of gene copies for SSU rDNA from H. volcanii, as estimated by 5′-nuclease assays using the ARCH1 set. Open circles, SSU rDNA in supercoiled plasmids; closed circles, genomic DNA; open squares: SSU rDNA in linearized plasmids.
FIG. 2
FIG. 2
Estimation of the percentage of H. volcanii SSU rDNAs in mixtures with DNA of strain R2A57. The x axis shows expected values calculated using various mixture proportions and the numbers of SSU rDNA copies of H. volcanii and strain R2A57 per microliter prior to mixing. The y axis shows the percentage of H. volcanii SSU rDNA copies estimated by 5′-nuclease assays using set ARCH1. Total SSU rDNA copies were estimated by 5′-nuclease assays using primer and probe sets ARCH1 and BACT1. The line represents a theoretical line assuming a 100% match in the values.
FIG. 3
FIG. 3
Estimation of the percentage of a Synechococcus sp. in mixtures with genomic DNA of strain R2A57. The x axis shows expected values calculated using various mixture proportions and the numbers of SSU rDNA copies of Synechococcus sp. strain WH7803 and strain R2A57 per microliter prior to mixing. The y axis shows the percentage of SSU rDNA from Synechococcus sp. strain WH7803 estimated by 5′-nuclease assays using set PHPICO. Total SSU rDNA was estimated by 5′-nuclease assays using set BACT2. Error bars indicate 1 standard deviation from the mean of triplicate reactions. The line represents a theoretical line assuming a 100% match in the values.
FIG. 4
FIG. 4
Depth profiles of Prochlorococcus and Synechococcus at station M1. Open circles, percentages of Prochlorococcus and Synechococcus SSU rDNAs measured by a 5′-nuclease assay using set PHPICO relative to bacterial SSU rDNA measured by a 5′-nuclease assay using set BACT2; closed squares, percentages of Prochlorococcus and Synechococcus cell numbers relative to total bacterioplankton cell numbers measured by flow cytometry.
FIG. 5
FIG. 5
Depth profiles of ratios between group I (GI) marine Archaea and group II (GII) marine Archaea at station M1. Open circles, ratios of SSU rRNAs measured by radiolabeled oligonucleotide dot blot hybridization; open squares, ratios of cell numbers per milliliter estimated using fluorescence-labeled polyribonucleotide probes and FISH; closed squares, ratios of SSU rDNA copies per nanogram of DNA estimated by 5′-nuclease assays using primer and probe sets ARCHGI and ARCHGII for group I and group II, respectively.
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References

    1. Amann R I, Krumholz L, Stahl D A. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 1990;172:762–770. - PMC - PubMed
    1. Amann R I, Ludwig W, Schleifer K H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995;59:143–169. - PMC - PubMed
    1. Ausubel F A, Brent R, Kingston R E, Moore D D, Smith J A, Seidman J G, Struhl K. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1988.
    1. 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. - PubMed
    1. Buck K R, Chavez F P, Campbell L. 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. 1996;10:283–298.

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