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Published on: June 11, 2016
Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community
Robert Brankatschk1, Natacha Bodenhausen, Josef Zeyer
1Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, Zurich, Switzerland. robert.brankatschk@env.ethz.ch
Applied and Environmental Microbiology
|April 12, 2012
Summary
The standard-curve method in real-time quantitative PCR (qPCR) can cause large errors due to differing amplification efficiencies. A new one-point calibration (OPC) method accurately quantifies microbial genes by correcting for these efficiency variations.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Real-time quantitative PCR (qPCR) is crucial for microbial community analysis.
- Absolute quantification often uses the standard-curve (SC) method, assuming equal amplification efficiency (E) between standards and samples.
- Discrepancies in E can lead to significant quantification errors.
Purpose of the Study:
- To evaluate the accuracy of the standard-curve (SC) method in qPCR for microbial gene quantification.
- To introduce and validate the one-point calibration (OPC) method as an alternative for accurate absolute quantification.
- To address the impact of varying amplification efficiencies on qPCR results.
Main Methods:
- Analysis of 19 bacterial strains and nine environmental samples using qPCR targeting nifH and 16S rRNA genes.
- Comparison of the standard-curve (SC) method with the proposed one-point calibration (OPC) method.
- Quantification of artificial template mixtures with known differing amplification efficiencies.
Main Results:
- Significant differences in amplification efficiency (E) were observed between different templates (bacterial strains and environmental samples).
- The SC method showed 3- to 5-fold deviations in quantifying nifH gene copy numbers in artificial mixtures.
- The OPC method demonstrated high accuracy in quantifying template mixtures and revealed significant differences compared to SC in environmental samples.
Conclusions:
- The assumption of equal amplification efficiency in the SC method can lead to substantial errors in qPCR-based microbial gene quantification.
- The one-point calibration (OPC) method effectively corrects for variations in amplification efficiency, providing more accurate absolute quantification.
- Accurate qPCR quantification, especially in complex microbial communities, necessitates methods that account for template-specific amplification efficiencies.
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