ArticleApplied and environmental microbiology2012
Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples.
Article in Applied and environmental microbiology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 91 papers.
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91 citing papers in PubMed.
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- Amplification Efficiency of Quantitative PCR Reactions is Improved by Addition of Non-Target DNA.Microbial ecology · 2026Article
- Bone from Healthy Individuals and Patients with CKD Expresses the Sodium-Glucose Co-transporter-2 (SGLT2).Calcified tissue international · 2026Article
- Culture, qPCR, and genome-based surveillance ofFrontiers in microbiology · 2026Article
- Improving biocide evaluation using propidium monoazide (PMA) viability staining technique.Scientific reports · 2025Article
- Analysis of qPCR Data: From PCR Efficiency to Absolute Target Quantity.International journal of molecular sciences · 2025Review
- Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR.Nature protocols · 2025Review
- Microbial diversity supports nitrification: insights from a full-scale anoxic/oxic wastewater treatment process.Applied and environmental microbiology · 2025Article
- Cooking-Derived Organic Compounds Shape Bacterial Communities on Household Surfaces.Environmental science & technology · 2025Article
- Counting soil microbial communities: the impact of qPCR platform and mastermix on accuracy and precision.FEMS microbiology ecology · 2025Article
- Quantification of Circulating Cell-Free DNA as a NETosis Marker in Trauma Patients with Type 2 Diabetes Mellitus.Methods and protocols · 2025Article
- Relative expression of pro-inflammatory cytokine genes in Holstein dairy cows naturally affected byIranian journal of veterinary research · 2025Article
- Assessment of absolute abundance in mother-infant gut microbiome using marine-sourced bacterial DNA spike-in and comparison with conventional quantification methods.Microbiome research reports · 2025Article
- Native Microalgae-Bacteria Consortia: A Sustainable Approach for Effective Urban Wastewater Bioremediation and Disinfection.Microorganisms · 2024Article
- Identification of commensal gut bacterial strains with lipogenic effects contributing to NAFLD in children.iScience · 2024Article
- Hsa-miR-194-5p and hsa-miR-195-5p are down-regulated expressed in high dysplasia HPV-positive Pap smear samples compared to normal cytology HPV-positive Pap smear samples.BMC infectious diseases · 2024Article
- Enhancing tumor-specific recognition of programmable synthetic bacterial consortium for precision therapy of colorectal cancer.NPJ biofilms and microbiomes · 2024Article
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4 authors.
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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.
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