Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2026:2969:235-247.
doi: 10.1007/978-1-0716-4767-7_14.

Digital Droplet PCR (ddPCR) for Absolute Quantification of 16S rRNA Copy Number in Metagenomic Data

Claudia Leoni #  1 , Marinella Marzano #  1 , Ermes Filomena  2 , Anna Maria D'Erchia  3
Affiliations

Digital Droplet PCR (ddPCR) for Absolute Quantification of 16S rRNA Copy Number in Metagenomic Data

Claudia Leoni et al. Methods Mol Biol. 2026.

Abstract

Digital Droplet PCR (ddPCR) is a quantitative PCR method that offers high sensitivity and accuracy in measuring the amount of nucleic acid in a sample, without the need of a standard curve. In ddPCR, a single sample is partitioned into up to 20,000 droplets, using the water-oil emulsion technology, and the amplification reaction occurs within each droplet using a fluorescent hydrolysis probe (Taqman) or a DNA-binding fluorescent dye. Following PCR, the emitted signals are individually measured in each droplet. Here, we describe a ddPCR optimized protocol for accurately quantifying the total copy number of the 16S rRNA gene in a metagenomic DNA sample. The protocol utilizes a primer pair, targeting the 16S V5-V6 hypervariable regions, in combination with a double-strand DNA-binding fluorescent dye.

Keywords: 16SrDNA; Absolute quantification; Metagenomics; Microbiome; ddPCR.

PubMed Disclaimer

References

    1. Verhaegen B, De Reu K, De Zutter L, Verstraete K, Heyndrickx M, Van Coillie E (2016) Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces. Toxins 8:157. https://doi.org/10.3390/toxins8050157 - DOI - PubMed - PMC
    1. Li F, Liu J, Maldonado-Gómez MX, Frese SA, Gänzle MG, Walter J (2024) Highly accurate and sensitive absolute quantification of bacterial strains in human fecal samples. Microbiome 12:168. https://doi.org/10.1186/s40168-024-01881-2 - DOI - PubMed - PMC
    1. Park C, Kim SB, Choi SH, Kim S (2021) Comparison of 16S rRNA gene based microbial profiling using five next-generation sequencers and various primers. Front Microbiol 12:715500. https://doi.org/10.3389/fmicb.2021.715500 - DOI - PubMed - PMC
    1. Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, Turner P, Parkhill J, Loman NJ, Walker AW (2014) Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol 12:87. https://doi.org/10.1186/s12915-014-0087-z - DOI - PubMed - PMC
    1. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41:e1–e1. https://doi.org/10.1093/nar/gks808 - DOI - PubMed

MeSH terms