Validation of pooled testing for SARS-CoV-2 using droplet digital PCR

PETRELI, MARIA VICTORIA; PEREZ, MARILINA; SESMA, JULIANA; HECKEL, SOFIA; PACINI, ANTONELLA; METZLER, PRISCILA; ADRIANI, NATALIA; PAREDES, FRANCO

Congreso; Reunión de Sociedades de Biociencias 2021 (SAIC SAI AAFE NANOMED.AR); 2021

142. (004) VALIDATION OF POOLED TESTING FOR SARSCoV-2 USING DROPLET DIGITAL PCRSofia Heckel, M. Victoria Petreli, Natalia Adriani, AntonellaPacini, Marilina Perez, Franco Paredes, Priscila Metzler, Juliana Sesma.Hospital Provincial de Rosario, Argentina; Instituto de inmunología clínica y experimental de Rosario (IDICER-CONICET); Facultad de Ciencias Médicas (FCM-UNR).The outbreak of COVID-19 has 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 thegeneration of 20,000 nanodrops per tube. This technology is rarelyused in clinical laboratories, due to its higher cost when comparedwith PCR. Cost could be reduced by using pooled testing. A negative test result indicates that all individuals in the pool are negativewhile a positive result indicates that at least one individual is positive. Pooled testing may be particularly useful to communities withlow prevalence of COVID-19. We proposed to validate the use ofddPCR to detect SARS-CoV-2 by pooling.Throat swab samples of 1000 patients were collected and soakedin 2mL saline. RNA extraction was done using automatic magneticextraction, columns extraction kits, and heat. Firstly, positive andnegative samples were identified with RT-PCR, pools of different sizes were designed and ddPCR was performed. Data was analyzedwith Quanta Soft analysis software v.1.7.4.0917 (Bio-Rad). Thisstudy was granted exception from bioethics committee approval asdeidentified remnants samples were used.We determined the specificity (we measured 100 negatives pools),the limit of detection (three independent octuplicates of the greatestdilution that it is positive) and the robustness of the method (the ability to withstand small but deliberate variations in method parametersby performing 20 repetitions changing the order of pooling and purification; and by measuring RNAs obtained using different extractionmethod). In the present work, we validated the use of pooled testingby combining up to 34 samples per pool.We hope that such implementation of a pool test for SARS-CoV-19would allow expanding current screening capacities, thereby enabling the expansion of detection in the community, as well as inclose organic groups.