Analysis of SARS-CoV-2 in wastewater for COVID-19 epidemiological control

The SARS-CoV-2 virus is a positive polarity single RNA virus belonging to the Coronavirus family. This virus is responsible for the COVID-19 disease, declared a pandemic in March 2020 by the World Health Organization. The main route of transmission of the virus is through the droplets that are released when sneezing, coughing, or talking; however, viruses have also been detected in the faecal matter of infected patients. 

So far there is no evidence that indicates that the SARS-CoV-2 virus is transmitted by this route (faecal – oral). However, analysing the level of virus in wastewater has proven to be advantageous in terms of epidemiological control of future outbreaks, since various studies seem to indicate that the detection of the virus in wastewater would occur 1 to 3 weeks before the increase in clinical cases of COVID-19 in the population.

SARS-CoV-2 detection protocol in wastewater: the concentration of the sample

The SARS-CoV-2 detection protocol in wastewater consists of 4 steps: sampling, concentration, RNA extraction and quantification. Briefly, between 500-1000 ml of samples are taken from the WWTPs (Wastewater Treatment Stations), and taken to the laboratory, where an aliquot of 200 ml is taken and the process begins. Samples, if possible, should be kept cold (4ºC).

The concentration of the sample is carried out by precipitation with aluminium chloride (AlCl3), which is the protocol described for concentrating enteric viruses from wastewater. This protocol alternates reagent addition steps, with pH adjustment steps and centrifugations. Specifically, two centrifugation steps are required: first at 1,700 xg for 20 minutes (200 ml in 250 ml tube), and then at 1,900 xg for 30 minutes (10 ml in 15 ml tube). As a result of this process, 2 ml of concentrated sample is obtained.

The refrigerated centrifuge Consul 22 R has a range of accessories that will allow us to work easily with both types of samples, keeping the working temperature stable thanks to its cooling system.

The RNA extraction from the sample

To extract the RNA from the sample, 100µL are taken from the concentrated sample, and the following procedure is started: cell lysis; separation of RNA from other macromolecules such as DNA, proteins and lipids; and concentration of the extracted RNA (9). The RNA extraction is carried out by ethanol precipitation, depositing the lysed sample in an extraction column coupled to a collecting tube of 2mL capacity, and centrifuging for 1 minute at 6,000 G each time a new reagent is added.

An elution is thus obtained with the extracted RNA, which will be quantified by RT-PCR (Real Time Reverse Transcription Polymerase Chain Reaction), following the manufacturer’s instructions in each case.

For the centrifugation of these samples, we can use a support minicentrifuge, like the Minicen with its rotor for microtubes, thus avoiding having to change the rotor in the high-capacity centrifuge in which we are working the other samples.

The detection of SARS-CoV-2 in wastewater seems to indicate the epidemiological evolution of COVID-19 in population. To carry out this protocol, the centrifugation of the sample is required for the concentration and RNA extraction steps. The combination of the Consul 22 R and Minicen centrifugals forms the perfect working package.