Detection and quantification of SARS-CoV-2 in wastewater samples

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is an animal pathogen that consists of:

1. An outer capsule, and
2. A single strand of ribonucleic acid (RNA) wrapped in a protein structure referred to as the nucleocapsid.

Detection of the virus requires targeting SARS-CoV-2 specific biomarkers, the specific genes encoding its structural features. The most common biomarkers for the SARS-CoV-2 virus are the N1 and N2 genes encoding the nucleocapsid protein (not to be confused with the capsule itself).

The analytical method for testing wastewater is very similar to individual clinical testing, however, in addition to confirming the presence or absence of the viral biomarkers, the method also quantifies the number of gene copies per unit volume. Analysis and quantification at Bureau Veritas is completed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).

SAMPLE COLLECTION AND PROCESSING

Based on literature data reported from studies looking at WBE for SARS-CoV-2, the most representative form of sampling wastewater influent is a composite, either time or flow weighted. Grab samples may also be used, but a clear understanding of population size and temporal influence is necessary. Samples collected should be immediately placed on ice prior to submission to the laboratory. RNA genetic material is significantly more prone to degradation compared to DNA because it is a single strand structure. Furthermore, the wastewater matrix contains other potential chemical agents that can further degrade target viral RNA. Although consensus has not been reached on whether the SARS-CoV-2 virus is still active at the point of influent collection, it is suspected that much of its capsule is degraded by then. Studies have indicated complete inactivation in wastewater between 2 to 4 days from collection.

Bureau Veritas’ method for RT-qPCR analysis of SARS-CoV-2 RNA requires a 250mL container supplied by
the laboratory (sterile and RNAse free). A 40mL aliquot is removed per sample for processing, extraction and analysis. Two other 40mL aliquots of the sample are temporarily stored for potential re-analysis (if required), one stored at 4°C and another at -80°C. Although a reference method is not yet available, best practice recommends samples be extracted within no more than 48 hours from collection. Once samples are extracted, the vials can be frozen for a short period of time, preventing the loss of RNA material.

Data accumulated so far on the analysis of SARS-CoV-2 in wastewater samples suggests the virus is hydrophobic and partitions well to solids. Bureau Veritas’ analytical method employs precipitation with polyethylene glycol (PEG) and refrigerated centrifugation to separate the solids during the sample preparation step.

SAMPLE ANALYSIS AND REPORTING

Prepared samples undergo RNA extraction, followed by 1-step RT-qPCR analysis. Quantitative PCR analysis requires double stranded genetic material, so the extracted RNA must be first converted into complementary DNA (cDNA) by the reverse transcriptase (RT) enzyme. Then the qPCR process amplifies the target genes and quantifies the copies in real time. The entire procedure is performed using one mix, hence the term “1-step”.

The SARS-CoV-2 target biomarker genes, the N1 and N2, are identified and quantified for each sample. In
addition, to report these quantitative results relative to the amount of faecal material captured by the sample, the pepper mild mottle virus (PMMoV) is also quantified. An internal positive control is also used to assess qPCR inhibition. An inhibited sample goes through an additional clean-up process before 1-step RT-qPCR analysis (N1, N2, PMMoV).

Analytical results will provide:

1. Indication of presence / absence of SARS-CoV2 biomarkers,
2. Quantified SARS-CoV-2 gene copies / mL (total N1 and N2),
3. Normalized SARS-CoV-2 gene copies / mL (in relation to the PMMoV), and
4. Sample inhibition status.