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

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.