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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 (E ) 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 thenifH and 16S rRNA genes. TheE values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in theirE 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 inE and was derived from the ΔΔCT method with correction forE , which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures fromGeobacter sulfurreducens (DSM 12127) andNostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in theirE values. While the SC method deviated from the expectednifH 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 inE between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples