Simple endpoint PCR assay using portable devices for Escherichia coli quantification in water samples

Area of Science:

• Environmental microbiology
• Molecular diagnostics
• Water quality assessment

Background:

• Quantitative detection of Escherichia coli (E. coli) is crucial for monitoring fecal contamination in water.
• Conventional quantitative real-time PCR (qPCR) methods are often expensive and inaccessible in resource-limited settings.
• There is a need for simple, portable, and cost-effective E. coli detection methods.

Purpose of the Study:

• To evaluate a simple endpoint PCR combined with SYBR Green I (SGI)-based colorimetric DNA detection (PCR-SGI) assay for E. coli quantification in water.
• To assess the feasibility of using portable devices for E. coli detection.
• To compare the performance of the PCR-SGI assay with conventional qPCR and colony-forming unit (CFU) counts.

Main Methods:

• Amplification of the E. coli 16S ribosomal RNA gene using endpoint PCR and a portable thermal cycler.
• Colorimetric DNA detection of PCR amplicons using SYBR Green I (SGI) and tablet-based image analysis.
• Integration of a modified DNA extraction protocol and portable devices (vortex mixer, portable centrifuge).

Main Results:

• The preliminary assay demonstrated a detection limit of 2.47 genome copies per reaction.
• The PCR-SGI assay successfully detected E. coli in various water samples, including domestic wastewater, lake, and river water.
• Results showed strong correlations with conventional qPCR (r=0.973) and CFU counts (r=0.917).

Conclusions:

• The endpoint PCR-SGI assay is a simple and effective method for quantifying E. coli in water samples.
• The assay's reliance on portable equipment makes it suitable for resource-limited settings.
• This method offers a viable alternative for rapid water quality assessment and public health monitoring.