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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Tracking Different States of Spiked Environmental DNA Using Multiplex Digital PCR Assays.

Julia Zöhrer1, Judith Ascher-Jenull2, Andreas O Wagner1

  • 1Department of Microbiology, Universität Innsbruck, Innsbruck, Austria.

Environmental Microbiology
|March 28, 2025
PubMed
Summary

This study introduces a new method to track intracellular (iDNA) and extracellular DNA (exDNA) from specific bacteria in environmental samples. The approach helps understand DNA extraction biases and differentiate DNA states for accurate microbial community analysis.

Keywords:
Bacillus subtilisEscherichia coliextracellular DNAintracellular DNAspike‐and‐recovery controls

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Area of Science:

  • Environmental microbiology
  • Molecular ecology
  • Genomics

Background:

  • Environmental DNA (eDNA) analysis is crucial for studying microbial communities.
  • eDNA exists in intracellular (iDNA) and extracellular (exDNA) forms, impacting extraction efficiency.
  • Current methods lack controls to differentiate eDNA states and bacterial origins.

Purpose of the Study:

  • To develop and validate spike-and-recovery controls for tracing iDNA and exDNA from specific bacterial strains.
  • To assess the influence of bacterial origin on DNA recovery in environmental samples.
  • To improve methodologies for eDNA extraction and state differentiation.

Main Methods:

  • Utilized single-gene deletion mutants of Escherichia coli and Bacillus subtilis as model organisms.
  • Developed unique primer/probe sets for absolute quantification of spiked iDNA and exDNA.
  • Employed multiplex digital PCR assays for precise DNA measurement.
  • Applied spike-and-recovery controls in diverse environmental matrices (soil, sediment, sludge, compost).

Main Results:

  • Successfully applied the proposed spike-and-recovery controls across various environmental samples.
  • Observed significant differences in iDNA recovery between E. coli and B. subtilis.
  • Demonstrated similar recovery rates for exDNA from both bacterial species, irrespective of origin.
  • Indicated that the environmental fate of DNA molecules is consistent across different bacterial sources.

Conclusions:

  • The developed method effectively differentiates iDNA and exDNA from specific bacterial sources in environmental samples.
  • The findings suggest that extracellular DNA behavior is independent of its bacterial origin.
  • This approach offers a valuable tool for addressing methodological ambiguities in eDNA extraction and analysis.
  • Future research can leverage this method to enhance the accuracy of microbial community assessments.