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Modern Molecular Taxonomy01:29

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Advancements in molecular biology have revolutionized the identification and characterization of bacteria, with multiple methods leveraging DNA sequencing for enhanced precision. As sequencing technologies improve and costs decline, these approaches are increasingly used in clinical, environmental, and evolutionary studies.Multilocus Sequence Typing (MLST) examines several housekeeping genes, essential chromosomal genes encoding cellular functions, to distinguish strains. Approximately...

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Optimizing Digital PCR Assays for Multiplexed Microbial Source Tracking in Surface Waters.

Shimul Ghosh1, Aaron Bivins2

  • 1Department of Civil & Environmental Engineering, Louisiana State University, Baton Rouge, LA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|October 10, 2025
PubMed
Summary

Digital PCR (dPCR) offers precise nucleic acid measurement for microbial source tracking (MST). Optimized triplex dPCR assays provide comparable results to single-plex methods, enhancing water quality analysis.

Keywords:
Cow M2Cow M3Digital PCREntero1aHF183/BacR287Microbial source trackingMultiplexPig2BacQIAcuityDeviceQIAcuityRum2Bac

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Area of Science:

  • Environmental microbiology
  • Molecular diagnostics
  • Water quality assessment

Background:

  • Digital PCR (dPCR) is a sensitive method for nucleic acid quantification.
  • Microbial source tracking (MST) relies on accurate detection of fecal indicator bacteria in water.
  • Multiplexing dPCR assays can improve efficiency for MST.

Purpose of the Study:

  • To optimize and validate two triplex digital PCR assays for microbial source tracking (MST).
  • To assess the performance of optimized triplex dPCR assays against single-plex assays.
  • To demonstrate the utility of the QIAcuity One system for multiplexed MST analysis.

Main Methods:

  • Empirical optimization of two triplex dPCR reactions (Triplex 1 and Triplex 2) on the QIAcuity One system.
  • Validation using a standard reference material (SRM 2917) across various concentrations.
  • Comparison of triplex assay results with single-plex assay results on environmental water samples.

Main Results:

  • Optimized triplex dPCR assays demonstrated comparable gene copy measurements to single-plex assays.
  • Consistent performance was observed for both standard reference materials and environmental water samples.
  • The developed triplex assays accurately measured MST markers in water samples.

Conclusions:

  • The optimized triplex dPCR assays are suitable for direct adoption in microbial source tracking (MST) on the QIAcuity system.
  • The described optimization protocol can guide the development of other multiplex dPCR assays.
  • Multiplexed dPCR offers an efficient and reliable approach for water quality monitoring.