Optimizing solid-based methods for SARS-CoV-2 detection in wastewater: addressing PCR inhibition and variant...

Area of Science:

• Environmental microbiology
• Molecular biology
• Public health

Background:

• Wastewater surveillance effectively monitors SARS-CoV-2 prevalence.
• Viral RNA dynamics in wastewater are not fully understood, impacting quantification accuracy.
• Existing solid-based RNA extraction methods require refinement for wastewater surveillance.

Purpose of the Study:

• To enhance a solid-based viral RNA extraction method for SARS-CoV-2 wastewater surveillance in Japan.
• To investigate and mitigate PCR inhibition affecting RNA quantification in wastewater solids.
• To assess the impact of SARS-CoV-2 variants on detection sensitivity.

Main Methods:

• Analysis of 84 RNA samples from wastewater influent and sludge using RT-qPCR.
• Spiking experiments with virus-like particles (VLPs) as processing controls.
• Evaluation of PCR inhibitor removal kits and simple dilution methods.

Main Results:

• PCR inhibition significantly impacted RNA samples from wastewater solid fractions.
• A commercially available inhibitor removal kit was ineffective; simple dilution mitigated inhibition.
• The Omicron variant emergence reduced the sensitivity of the CDC N1 RT-qPCR assay.

Conclusions:

• The study improved a solid-based RNA extraction method by addressing PCR inhibition and variant effects.
• Regular evaluation and quality control are crucial for refining wastewater surveillance methodologies.
• Adaptable methods are essential for effective management of evolving viral outbreaks.