Wastewater Surveillance Methodology

Kishwaukee Water Reclamation District places composite sampling pumps at three strategic locations in its service area, including the treatment plant, which receives inflow from its service area in DeKalb. The pumps are programmed to take a small volume of wastewater at specific time intervals over a 24-hour period; they are combined into a 1-liter composite sample, which represents content that has flowed past the access point over the span of a day. The composite wastewater samples are delivered to NIU’s wastewater lab three times per week, where it is tested for the SARS-CoV-2 virus.

SARS-CoV-2 viruses are shed in the stool of infected people. After Pasteurization to sterilize it, the wastewater sample is measured for its suspended biological content, termed “biomatrix”, by percent light transmittance using a spectrophotometer.

100 ml of wastewater (about 10% of the sample) is strained over a 60 mesh screen to remove large aggregates, then vacuum filtered over a small pore size membrane (a round special paper disc) that retains biomatrix. RNA is extracted from the biomatrix captured and concentrated on the membrane. This process is repeated three times for each sample.

RNA in the wastewater is purified during an extensive two-day process, and measured for purity and content.

Reactions are set up where the RNA is first converted to DNA by a process called Reverse Transcription (RT) and then amplified by the Polymerase Chain Reactions (PCR). The tests are run in special plates designed to be read in a dedicated instrument. The entire process is referred to as the RT-qPCR reaction.

Samples are measured for two SARS-CoV-2 genes (N1 and N2 nucleocapsid proteins). The instrument uses known N1 and N2 gene standards run in the same plate to quantify the amount of SARS-CoV-2 RNA in the sample.