Sample Collection and Laboratory Methods

Sample collection is done by the participating utilities.  This program is reliant upon our utility partners for the proper collection, handling, and storage of the sample until the sample is picked up for transport to the laboratory, DCLS.  

There are two main types of samples that are collected:  grab samples and composite samples.  

  • Grab samples are a single sample collected at a moment in time. This type of sample is representative of the wastewater flowing into the treatment plant at that moment the sample is collected.
  • Composite samples are individual samples collected over a 24-hour time period and then combined to form a single sample.  The samples can be collected based on a set time (for example, every 10 minutes) or depending on how much flow enters the wastewater treatment plant.  This type of sample is representative of the whole day of flow into the wastewater treatment plant.

The utility provides additional information (metadata) that accompanies the sample such as time of sample collection, the flow for the day of collection, and the sampling point.  The samples are all collected at the entrance to the treatment plant before any significant processing occurs.

Samples are shipped to DCLS for concentration, extraction, and droplet digital polymerase chain reaction (ddPCR) analysis. Prior to the week of November 15, 2021, extracted samples were shipped to the NELAP-Accredited laboratory at Hampton Roads Sanitation District (HRSD) for SARS-CoV-2 analysis via ddPCR. The gene target presented in this report is ddCoV_N, a modified version of the N2 gene for ddPCR wastewater analysis[1]. Quantification results for which amplification was observed in the no-template controls (NTC) are removed from analysis. The limit of detection for the current version of the laboratory protocol (v2.0) is 6,200 copies/L of wastewater. All concentrations below the limit of detection are assigned a value equal to one half the limit of detection. 


Lab Protocol Version Limit of Detection (LOD) Laboratory Responsibilities
v2.0 6,200 Concentration, extraction, and ddPCR analysis: DCLS
v1.0 1,350 Concentration and extraction: DCLS

Droplet digital polymerase chain reaction (ddPCR) analysis: HRSD


Viral load is calculated based on Equation 1:

(1) Viral load (copies/day) = Viral conc. (copies/L)∗Flow rate (MGD)∗(3785411.8 L/d)/(1 MGD)


Percentiles are calculated on a per-site basis by viral load, and then binned into quintiles (i.e., 0-19.9th, 20-39.9th, 40-59.9th, 60-79.9th, 80-100th). Site-specific percentiles provide a metric to compare the current week’s data to the site’s historical data, which allows for color visualization of when each site experienced its highest versus lowest loads.


[1] Gonzalez, R., Larson, A., Thompson, H., Carter, E., & Cassi, X. F. (2021). Redesigning SARS-CoV-2 clinical RT-qPCR assays for wastewater RT-ddPCR.