VALIDATION OF POOLED TESTING FOR SARS-CoV-2 USING DROPLET DIGITAL PCR

The outbreak of COVID-19 has spread around the world and become a public health emergency. Viral nucleic acid detection by reverse transcription PCR (RT-PCR) is the gold standard method for diagnosis of COVID-19. Droplet digital PCR (ddPCR) is a highly sensitive PCR technology based on the generation of 20,000 nanodrops per tube. This technology is rarely used in clinical laboratories, due to its higher cost when compared with PCR. As the use of pooled testing greatly reduces the costs, we proposed to use ddPCR to detect SARS-CoV-2 of pooled samples. A negative test result indicates that all individuals in the pool are negative while a positive result indicates that at least one individual within the pool is positive. Pooled testing may be particularly useful to communities with low prevalence of COVID-19. For example, detection in the bubbles of workplaces, schools and sport competitions would allow to isolate a positive bubble and stop the widespread of the virus in that community. In the present work, we validated the use of pooled testing by combining up to 34 samples per pool. In order to do it, we determined the specificity (we measured 100 negatives pools), the limit of detection (three independent octuplicates of the greatest dilution that it is positive) and the robustness of the method (the ability to withstand small but deliberate variations in method parameters by performing 20 repetitions changing the order of pooling and purification; and by measuring RNAs obtained using different extraction method: magnetic beads, columns and heat).