Rapid and sensitive detection of MS2 coliphages in wastewater samples by quantitative reverse transcriptase PCR

Area of Science:

• Environmental microbiology
• Virology
• Molecular biology

Background:

• Coliphage MS2 serves as a surrogate for pathogenic viruses in wastewater research.
• Accurate quantification of viral indicators is essential for wastewater treatment monitoring.

Purpose of the Study:

• To develop and validate a rapid quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay for MS2 coliphage detection in treated wastewater.
• To compare the performance of the developed qRT-PCR assay with the traditional plaque assay.

Main Methods:

• Development of a SYBR Green-based qRT-PCR assay with an internal control for inhibitor detection.
• Validation using artificially contaminated water samples.
• Application to naturally contaminated wastewater samples from a treatment plant and comparison with plaque assay results.

Main Results:

• The qRT-PCR assay demonstrated a wide dynamic range (8 logs) with high linearity (R²=0.999).
• The method achieved a limit of detection of 4x10^2 genome equivalents/100 ml.
• The qRT-PCR method detected viral counts approximately 1.5 orders of magnitude higher than the plaque assay.
• The entire assay, including sample processing, was completed within 4 hours.

Conclusions:

• The developed qRT-PCR assay is a rapid, sensitive, and accurate method for quantifying MS2 coliphages in wastewater.
• This method provides a significant improvement over traditional plaque assays, offering higher sensitivity and speed.
• The assay's rapid turnaround time makes it suitable for single-day wastewater analysis and monitoring.