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Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens.

Angela Sun1, Jo-Ann L Stanton2, Peter L Bergquist1,3

  • 1Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.

Microorganisms
|November 27, 2021
PubMed
Summary

A new workflow rapidly quantifies major waterborne pathogens like Cryptosporidium and E. coli. This method uses filtration and enzyme-based DNA extraction for sensitive detection in tap water.

Keywords:
Campylobacter jejuniCryptosporidium parvumDNA extractionEscherichia coliGiardia lambliaenumerationfiltrationpotable waterqPCR quantificationwaterborne pathogen

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Area of Science:

  • Environmental microbiology
  • Water quality analysis
  • Molecular diagnostics

Background:

  • Accurate detection of waterborne pathogens is crucial for public health.
  • Conventional methods for pathogen quantification are often slow and labor-intensive.
  • Existing methods struggle with large sample volumes and sample inhibition from tap water matrices.

Purpose of the Study:

  • To develop a rapid, sensitive, and universal workflow for quantifying key waterborne pathogens.
  • To establish a method suitable for large tap water volumes (up to 100 mL).
  • To overcome inhibition issues caused by tap water particulate matter.

Main Methods:

  • A single-step workflow combining polycarbonate filtration and enzyme-based DNA extraction (prepGEM).
  • Direct quantitative Polymerase Chain Reaction (qPCR) on extracted DNA.
  • On-filter treatment with phosphoric acid to remove inhibitors.

Main Results:

  • Achieved low limits of quantification: 4 oocysts (Cryptosporidium), 12 cysts (Giardia), 2 cells (C. jejuni), 19 cells (E. coli) per reaction.
  • Workflow requires only 20 min incubation and a 100 µL reaction mix.
  • Phosphoric acid wash effectively removed tap water inhibition, restoring quantification efficiency.
  • Achieved >92% quantification efficiency for all four target pathogens.

Conclusions:

  • The established workflow provides a rapid, portable, and cost-effective solution for water monitoring.
  • This method enables sensitive and accurate quantification of multiple leading waterborne pathogens.
  • The workflow bypasses traditional culture methods, significantly reducing analysis time.