DIGICOVID_VAR - Development of a digital PCR method for accurate detection and quantification of SARS-CoV-2 and its variants

This project develops a digital multiplex PCR method targeting specific genomic regions of SARS-CoV-2 variants circulating in the EU in order to allow a more accurate detection and quantification of SARS-CoV-2 variants in humans as well as in environmental samples (like wastewater). This project is a follow-up of a first project named DIGICOVID and in link with the surveillance of wastewater. In the DIGICOVID project, Sciensano developed a new digital multiplex PCR (ddPCR) method for the detection and quantification of SARS-CoV-2. This follow-up project focuses on the development of (a) multiplex ddPCR method(s) targeting the variants in humans as well as in environmental samples. Sciensano develops and validates the ddPCR on purified RNA controls and then tested on a selection of human and wastewater samples.

Viruses change constantly through mutations, and new variants of a virus are expected to occur over time. Certain mutations may affect its spreading (e.g. VUI 202012/01 SARS-CoV-2 variants) or cause more severe illness.

Therefore, it is important to develop methods to detect rapidly these variants. Up to now, whole genome sequencing (WGS) seemed to be the most appropriate technology to detect variants characterised by several genetic modifications (mutations, insertions, deletions). However, WGS is more expensive and is more complicated to implement than PCR methods that are routinely used in diagnostics to detect SARS-CoV-2. Moreover, not all the diagnostic labs are equipped to generate and analyse WGS data. In addition, sometimes WGS cannot be applied on a sample because the nucleic acid concentration is too low or of bad quality. 

Therefore, Sciensano proposes to use PCR based methods as a first line screening, to detect a mutation representative of the variants (often present in the variants and not often present in the other SARS-CoV-2 genomes). This method has several advantages:

• It allows a fast identification of suspected samples (after a few hours). WGS will be used in a second stage to confirm the variants. 
• The PCR method(s) used to detect the variant(s) are developed similarly to the general method previously developed in the DIGICOVID project to detect all the SARS-CoV-2 strains (all variants) in order to be able to use these methods together. 
• The selected technology will be digital PCR that presents several advantages like a high sensitivity, analysing difficult samples that often cause inhibition in PCRs (e.g. environmental samples like wastewater or faecal samples) and absolute quantification of the virus. 
• The use of the universal ddPCR for SARS-CoV-2 genomes in combination with the specific multiplex ddPCR methods targeting SARS-CoV-2 genome variant(s) allows to quantify the percentage  of variants in regard to the total SARS-CoV-2 virus in a samples.