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Background on the waste water monitoring programme

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During the project implementation period (01.01.2022 - 30.09.2023) a fully equipped molecular biology laboratory was developed for monitoring Covid 19 and its variants in wastewater and from 19.07.2023 weekly sampling from 7 strategically selected points in Bucharest and analysis of the samples was performed.

The monitoring of SARS-CoV-2 in wastewater involves the following steps: weekly collection of samples from the 7 established points in Bucharest, reception of samples, their primary processing, RNA extraction, amplification of extracts, data interpretation, sequencing of positive samples, and finally interpretation and reporting of the data obtained.

Once the samples are taken from the field and received, they are subjected to primary preparation. The step involves aliquoting the samples in smaller volumes into 50 mL tubes, to which the BCoV process control is added. Subsequently, after a 10-minute centrifugation, 14 mL of these are transferred to Vivaspin 20 (Millipore) membrane filter tubes and centrifuged for 15 minutes for ultrafiltration. Following collection of the wastewater concentrates in Eppendorf tubes (2 mL), the processed samples can be prepared for extraction and loaded into the QIAcube automatic extractor. For the viral RNA amplification step, the extracts obtained are coupled with an enzyme mix for the PCR reaction to which coronavirus-specific probes for the BCoV, N1 and N2 genes are added. Thus, the mix can be loaded into the QIAcuity 26K 24-well plate and inserted into the QIAcuity Digital PCR for analysis. If the Digital PCR results indicate positive samples, they can be sequenced using the Illumina MiSeq machine to determine possible mutations in the SARS-CoV-2 genome.

The results of the sample processing will be sent to the Ministry of Environment, Water and Forests in order to ensure Romania's reporting as a Member State to the European Commission.

 

Future perspectives

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Following the implementation of the project "Support to Member States for the establishment of national systems, local collection points and digital infrastructure for the monitoring of Covid 19 and its variants in waste water", it is proposed to extend the research area to the entire territory of Romania.