Comparison of RT-qPCR and RT-ddPCR on Assessing Model Viruses in Wastewater

Area of Science:

• Environmental microbiology
• Molecular biology
• Water quality analysis

Background:

• Quantifying viral loads in wastewater is crucial for public health and environmental monitoring.
• Polymerase chain reaction (PCR)-based methods, including RT-qPCR and RT-ddPCR, are commonly used for viral quantification.
• Wastewater matrices present challenges due to varying physical and chemical characteristics and potential inhibitors.

Purpose of the Study:

• To evaluate and compare the performance of reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR) for viral load quantification in wastewater.
• To assess the impact of RNA extraction and wastewater matrix composition on the accuracy and sensitivity of both methods.
• To determine the optimal method for precise viral monitoring in diverse wastewater treatment stages.

Main Methods:

• A model virus, Pseudomonas phage Φ6 (Phi6), was spiked into synthetic and real wastewater samples collected from multiple treatment stages.
• Performance of RT-qPCR and RT-ddPCR was compared with and without prior RNA extraction from wastewater samples.
• Viral quantification accuracy, sensitivity, and resilience to inhibitors and water quality variations were assessed.

Main Results:

• Both RT-qPCR and RT-ddPCR showed comparable performance for high and medium viral loads.
• RT-ddPCR exhibited significantly higher sensitivity for detecting low viral loads, outperforming RT-qPCR in trace level detection.
• RT-ddPCR with RNA extraction demonstrated improved performance against inhibitors, but was more affected by water quality without extraction.

Conclusions:

• RT-ddPCR is a more sensitive method for quantifying low viral loads in wastewater compared to RT-qPCR.
• RNA extraction enhances RT-ddPCR's resilience to wastewater inhibitors, making it suitable for precise viral monitoring.
• Despite higher costs and processing time, RT-ddPCR's superior sensitivity and robustness justify its use in critical wastewater applications.