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Quantification of Circular RNAs Using Digital Droplet PCR
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Variably improved microbial source tracking with digital droplet PCR.

Jean Pierre Nshimyimana1, Mercedes C Cruz2, Stefan Wuertz3

  • 1School of Civil and Environmental Engineering, Nanyang Technological University (NTU), 50 Nanyang Avenue, Singapore, 639798, Singapore; Singapore Centre for Environmental Life Sciences Engineering, NTU, 60 Nanyang Dr., Singapore, 637551, Singapore; Department of Civil and Environmental Engineering, Massachusetts Institute of Technology (MIT), 77 Massachusetts Avenue, Cambridge, MA, 02139, USA.

Water Research
|May 17, 2019
PubMed
Summary
This summary is machine-generated.

Digital droplet PCR (ddPCR) offers improved specificity for detecting human-associated Bacteroidales markers, enhancing microbial source tracking accuracy. While qPCR shows higher sensitivity in environmental samples, ddPCR provides greater reproducibility for fecal samples.

Keywords:
Digital droplet PCRGenetic markersMicrobial source trackingQuantitative PCR

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Area of Science:

  • Environmental microbiology
  • Molecular biology
  • Water quality analysis

Background:

  • Accurate microbial source tracking is crucial for assessing water quality and public health.
  • Human-associated Bacteroidales markers are commonly used, but their detection methods require optimization.
  • Quantitative PCR (qPCR) is a standard method, but digital droplet PCR (ddPCR) may offer enhanced performance.

Purpose of the Study:

  • To evaluate the sensitivity and specificity of ddPCR compared to qPCR for human-associated Bacteroidales markers (BacHum and B. theta).
  • To assess marker quantification in various sample types, including environmental and fecal composites.
  • To determine the suitability of ddPCR for microbial source tracking applications.

Main Methods:

  • Human markers BacHum and B. theta were quantified using both qPCR and ddPCR platforms from the same manufacturer.
  • A total of 180 diverse samples were analyzed, including human and animal feces, sewage, and environmental water.
  • Assay performance metrics such as sensitivity, specificity, accuracy, and reproducibility were compared between platforms.

Main Results:

  • ddPCR demonstrated improved specificity for BacHum detection in fecal samples (0.88 vs. 0.58 for qPCR) and increased accuracy (0.94 vs. 0.77).
  • Both platforms showed high sensitivity (0.85-1.00) in sewage and human stool, with qPCR exhibiting higher sensitivity in environmental samples.
  • ddPCR provided greater reproducibility in fecal composites, while marker concentrations were consistently lower than qPCR measurements.

Conclusions:

  • ddPCR is a valuable tool for microbial source tracking, offering enhanced specificity and reproducibility for certain markers and sample types.
  • Differences in assay performance may relate to target gene characteristics (copy number, gene type).
  • Cost-effectiveness and assay-specific performance should be considered when selecting between ddPCR and qPCR.