Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples

Abstract

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 the nifH and 16S rRNA genes. The E 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 E 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 E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH 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 E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

Fig 1

(A) Schematic amplification plot of log fluorescence increase over qPCR cycles, indicating the slope to estimate E_fi by linear regression. (B) Schematic standard curve of a dilution series, plotting C_T values over log template concentrations. The slope is used to estimate E_ds.

Fig 2

Variability of qPCR efficiency, E_fi, of bacterial strains (left) and environmental samples (right) targeting nifH and the 16S rRNA gene, estimated from the fluorescence increase. The error bars give the standard deviation of four replicates. The letters indicate significance groups by ANOVA (P < 0.01) tested for each panel individually. n.d., no amplification detected.

Fig 3

Quantification of a hypothetical sample (E_sample = 1.99; C_T = 21) by the SC and OPC methods using M. trichosporium (E_standard = 1.70) as a standard.

Fig 4

qPCR efficiency, E_fi, of defined mixtures of G. sulfurreducens and N. commune templates amplified with nifHF/nifHR primers. The values are the means of three replicates; the error bars indicate the standard deviations.

Fig 5

Copy numbers of defined mixtures of G. sulfurreducens (G. sul) and N. commune (N. com) templates estimated using the SC and OPC methods that were calibrated using both strains. The assays used nifHF/nifHR primers. The dashed lines give the expected copy numbers. The error bars give the standard deviations.

Fig 6

Comparison of the OPC and SC methods for quantifying nifH gene copy numbers in environmental samples using nifHF/nifHR primers. The error bars give the standard deviations. The asterisks mark significant differences between the two methods by the Mann-Whitney rank sum test (P < 0.01).