THE NOVELTY OF IDENTIFYING SARS-CoV2 VARIANTS IN POOLED SAMPLES USING DROPLET DIGITAL PCR

HECKEL SOFIA; PAREDES FRANCO; ADRIANI NATALIA; IBARRA GUADALUPE; PEREZ MARILINA; PACINI ANTONELLA; PETRELI MARIA VICTORIA; SESMA JULIANA

Mar del plata

Congreso; Reunión anual de sociedades de biociencias SAIC.SAI&FAIC.SAFIS; 2022

Considering that new SARS-CoV-2 variants continue emerging and monitoring their circulation through genomic surveillance remains important, our aim is to demonstrate the feasibility of differentiating SARS-CoV-2 variants in grouped positive samples. One of the most important applications of droplet digital PCR (ddPCR) is rare mutation detection. Here the challenge is the discrimination between two highly similar sequences: the variant present at a very low frequency in a vast pool of wild-type (WT). This is possible due to the partition of the sample into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Previously, we validated the use of ddPCR for testing SARS-CoV-2 in pooled samples by combining up to 34 samples per pool. Now, we are extending ddPCR use to identify SARS-CoV-2 variants in a pool of SARS-CoV-2 positive samples. A negative test result will indicate that all individuals in the pool are WT for that specific mutation while a positive result will indicate that at least one individual within the pool carries that mutation. Test were performed from July 2021 to March 2022 with de-identified samples. SARS-CoV-2 positive samples were identify by RT-PCR and later variants were genotyped by RT-PCR. Pools of different sizes (1 variant in a group of 5, 10 or 15 WT samples) were designed and ddPCR was performed. Data was analyzed with Quanta Soft analysis software (Bio-Rad). In order to detect two SARS-CoV-2 variants of concern: Delta and Omicron, the TaqMan SARS-CoV-2 Mutation Panel from Thermo Fisher was used for both RT- and ddPCR. In the present work, we demonstrated that for pools of up to 10 samples, ddPCR present 100% sensitivity (n=24) and >98% specificity (n=50) when analyzing 3 different probes for delta and/or omicron SARS-CoV-2 variants. Therefore, we reported the novel diagnostic technology of SARS-CoV-2 pool genotyping by ddPCR to achieve quick results (< 24 hs), high testing throughput and low costs.