Analysis Methods

After collection, samples should be refrigerated at 4ºC and ideally processed within 24 hours for optimal results. Both liquid wastewater samples and primary sludge samples should be well-mixed (either by inverting or mechanically mixing) before testing. Samples can be clarified using filters or centrifugation to remove any large solids, which can help with subsequent filtration and concentration steps but may result in RNA loss.

Sample Preparation.

Sample Concentration.

The first step in testing wastewater is sample concentration. Multiple methods are available, including electrostatic filters, ultrafiltration, polyethylene glycol precipitation, and ultracentrifugation.

  • Electrostatic filters with magnesium chloride pre-treatment and/or acidification: Concentrating SARS-CoV-2 RNA in a sample can be achieved by pre-treating the sample using acidification and/or magnesium chloride. Acidification enhances RNA recovery when using an electronegative filter, while magnesium chloride promotes RNA folding and condensation, aiding isolation (Cashdollar & Wymer, 2013). The sample is then passed through a ~5µm pore-size electronegative filter, concentrating the viral RNA.

  • Ultrafiltration/centrifugal filtration: SARS-CoV-2 RNA can also be isolated using ultracentrifugation or centrifugal filtration. Samples are centrifuged at refrigeration temperature to separate the heavier solids from the lighter liquid portion containing the RNA (the supernatant). The supernatant is then centrifuged faster and repeatedly to isolate the RNA (Ahmed et al., 2020).

  • Polyethylene glycol precipitation: Viral RNA concentration from a water matrix can be achieved using polyethylene glycol (PEG) precipitation. The sample is repeatedly centrifuged and agitated as in ultrafiltration, with the addition of PEG. PEG precipitates out of the solution, forming a pellet at the bottom of the centrifuge tube. The remaining liquid contains concentrated viral RNA. This method can isolate viral RNA from both solid and liquid phases and process large volumes of wastewater simultaneously (Ahmed et al., 2020).

  • Ultracentrifugation: The sample is first centrifuged at high speeds to extract RNA using ultracentrifugation. The supernatant is carefully removed, and the solid pellet formed at the bottom is further treated with a buffer solution, incubation on ice, neutralization, and clarification to isolate the viral RNA. This method concentrates viruses from both solid and liquid phases (Ahmed et al., 2020).

RNA Extraction.

Following concentration using one of the above-mentioned methods, the RNA is extracted from the suspension and purified to eliminate unwanted compounds. Like other environmental samples, wastewater samples are prone to containing unwanted compounds even after the concentration process. Extraction procedures aim to produce highly purified viral RNA extracts. To prevent RNA degradation, multiple freeze-thaw cycles are avoided by aliquoting samples into separate tubes and storing them at deep freezing temperatures ( ≤ –70ºC).

RNA Measurement.

Once viral RNA is extracted, it can be quantified using various detection methods. Polymerase Chain Reaction (PCR) is the most common amplification and detection method, with several types of PCR methods applicable under different conditions. These PCR tests are quite similar to individual-level diagnostic tests for COVID-19.

  • RT-qPCR (reverse transcription-quantitative polymerase chain reaction): RT-qPCR is a real-time RNA amplification, detection, and quantification method used in wastewater surveillance to detect and quantify viral mRNA. Extracted RNA from a sample is reverse-transcribed to DNA using an enzyme, and additional DNA fragments complementary to parts of the viral genome are added. If viral RNA is present, these complementary DNA fragments will bind to it. The mixture is placed into an RT-qPCR machine, which cycles through various temperatures to trigger chemical reactions that produce identical copies of parts of the transcribed DNA. This process amplifies the transcribed DNA, and the DNA fragments are tagged using fluorescent dye for detection by the machine's computer.

  • RT-ddPCR (reverse transcription droplet digital polymerase chain reaction): RT-ddPCR, similar to RT-qPCR, amplifies and detects RNA. It is performed using individual sample droplets, bypassing much of the dilution process required with other PCR analyses. The sample is partitioned into individual droplets in one step. One-step RT-ddPCR is beneficial for wastewater surveillance of SARS-CoV-2 because it uses individual sample droplets. Reverse transcription occurs in those droplets rather than in bulk solution, reducing reverse transcription inhibition and decreasing the chance of error.

Laboratory Controls.

Various control methods are employed in laboratory testing to ensure samples are uncontaminated and testing protocols are working correctly.

  • Matrix recovery control: Matrix recovery control assesses the amount of viral RNA lost during sample testing and processing. It should be included with each sample group to account for fluctuations in wastewater conditions and composition. Matrix recovery control is essential when wastewater conditions, sample collection methods, or laboratory testing protocols change.

  • Human fecal indicator: A human fecal indicator (or human fecal normalization) measures organisms specific to human feces in wastewater samples to estimate the sample's human fecal content. Viral molecular targets include Pepper Mild Mottle virus and crAssphage.

  • Quantitative measurement controls: Quantitative measurement controls depend on the RNA measurement method used. For RT-qPCR, a calibration curve can be derived from a control with a known RNA concentration, which can be used to control experimental samples. For RT-ddPCR, a control sample with a known RNA quantity can be included in each test. Since these tests detect viral RNA presence, RNA controls are preferred over DNA controls. Control samples should avoid multiple freeze-thaw cycles like experimental samples and be stored at or below -70ºC.

  • Inhibition assessment: Inhibition assessment determines whether the RNA quantification processes (both reverse transcription and polymerase chain reaction) are performing according to the protocol.