Wastewater monitoring programs are essential for communities to identify viral outbreaks and inform public health responses. However, traditional wastewater testing methods leave room for inhibitors to interfere with molecular diagnostic methods like qPCR, often giving public health officials an inaccurate estimation of virus levels in their community.
To fix this, scientists need the right tools to propel their virus detection programs forward, giving them faster access to data that is more accurate so public health officials can act quickly. With the help of the FluidPrep™ Concentrating Pipette System by InnovaPrep, researchers and public officials from the Northern Ireland National Monitoring Program were able to do just that.
The scientists chose the CP Select™ Concentrating Pipette for its ability to concentrate wastewater for rapid detection of viruses, even at low levels. With their new launchpad for virus detection, they 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 team processed the wastewater influent samples following a modified version of InnovaPrep's published protocol. First, they added Tween 20 to the raw wastewater, followed by centrifugation at 4000 x g for 10 minutes at 4°C. The supernatant (50 mL) was concentrated using the CP Select™ Concentrating Pipette with ultrafilter pipette tips, then eluted twice with InnovaPrep’s FluidPrep™ Elution Buffer.
To reduce inhibition, the Northern Ireland Monitoring Program tested neat and diluted samples using various PCR template dilutions and stabilizer concentrations including RNA/DNA Shield by Zymo and RNALater by Sigma Aldrich. They performed sample extraction with the Roche MagNA Pure kit, and conducted PCR testing on a Roche LightCycler 480. Finally, whole genome sequencing was completed using an Illumina MiSeq with ARTIC protocols.
Because the CP Select™ gave scientists a concentrated sample, results showed that both template dilution and the addition of stabilizing agents to wastewater samples improved SARS-CoV-2 detection by RT-qPCR.
The InnovaPrep Concentrating Pipette tool and sampling method was the launch pad that allowed for a faster and more accurate testing ground to detect viruses in the wastewater samples. Specifically, researchers found a 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 Full Test Results in Letters in Applied Microbiology
Improved recovery of SARS-CoV-2 from wastewater through application of RNA and DNA stabilizing agents. Bell, et al. Letters in Applied Microbiology, 2023
