A major advantage for wastewater monitoring programs is the ability to effectively sequence and identify new variants, even at low community levels, aiding in prompt and accurate public health responses. However, wastewater is a complex mixture containing inhibitors that can interfere with molecular diagnostic methods like qPCR, often leading to underestimation of virus levels within communities.
The Concentrating Pipette’ was selected for the Northern Ireland National Monitoring Program for its ability to concentrate wastewater for rapid detection of viruses even at low levels. Scientists there have further enhanced the method by remarkably diminishing PCR inhibition post-concentration through dilution of the PCR template and adding stabilizing agents to the concentrated sample prior to analysis.
The wastewater influent samples are processed following a modified version of InnovaPrep's published protocol. First, Tween 20 is added to the raw wastewater, followed by centrifugation at 4000 x g for 10 minutes at 4°C. The supernatant (50 mL) is concentrated using the Concentrating Pipette (CP) with ultrafilter pipette tips, then eluted twice with InnovaPrep’s Tris Elution Buffer.
To reduce inhibition, various PCR template dilutions and stabilizer concentrations (including RNA/DNA Shield by Zymo and RNALater by Sigma Aldrich) were tested on both neat and diluted samples. Sample extraction was performed with the MagNA Pure kit (Roche), with PCR conducted on a LightCycler 480 (Roche). Whole genome sequencing was completed using an Illumina MiSeq with ARTIC protocols.
Results showed that both template dilution and the addition of stabilizing agents to wastewater samples concentrated by the CP method improved SARS-CoV-2 detection by RT-qPCR. Specifically, DNA/RNA Shield at 0.5x concentration enhanced detection in undiluted samples by 4.2 Cp, while RNALater at 0.5x improved detection by 3.7 Cp. For sequencing, DNA/RNA Shield produced optimal results in samples diluted 1.5x, while RNALater performed best at a 0.5x dilution.
Read The Publication
Improved recovery of SARS-CoV-2 from wastewater through application of RNA and DNA stabilising agents. Bell, et al. Letters in Applied Microbiology,2023
