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Medientyp:
E-Artikel
Titel:
Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples
Beteiligte:
Brankatschk, Robert;
Bodenhausen, Natacha;
Zeyer, Josef;
Bürgmann, Helmut
Erschienen:
American Society for Microbiology, 2012
Erschienen in:Applied and Environmental Microbiology
Sprache:
Englisch
DOI:
10.1128/aem.07878-11
ISSN:
0099-2240;
1098-5336
Entstehung:
Anmerkungen:
Beschreibung:
<jats:title>ABSTRACT</jats:title>
<jats:p>
Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (
<jats:italic>E</jats:italic>
) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the
<jats:italic>nifH</jats:italic>
and 16S rRNA genes. The
<jats:italic>E</jats:italic>
values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their
<jats:italic>E</jats:italic>
values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in
<jats:italic>E</jats:italic>
and was derived from the ΔΔ
<jats:italic>
C
<jats:sub>T</jats:sub>
</jats:italic>
method with correction for
<jats:italic>E</jats:italic>
, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from
<jats:named-content content-type="genus-species">Geobacter sulfurreducens</jats:named-content>
(DSM 12127) and
<jats:named-content content-type="genus-species">Nostoc commune</jats:named-content>
(Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their
<jats:italic>E</jats:italic>
values. While the SC method deviated from the expected
<jats:italic>nifH</jats:italic>
gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in
<jats:italic>E</jats:italic>
between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.
</jats:p>