Pooled RNA extraction and PCR assay for efficient SARS-CoV-2 detection

Roni Ben-Ami; Agnes Klochendler; Matan Seidel; Tal Sido; Ori Gurel-Gurevich; Moran Yassour; Eran Meshorer; Gil Benedek; Irit Fogel; Esther Oiknine-Djian; Asaf Gertler; Zeev Rotstein; Bruno Lavi; Yuval Dor; Dana G Wolf; Maayan Salton; Yotam Drier

A key requirement of pooled RNA extraction and RT-PCR tests is to retain sufficient sensitivity. In our RT-PCR assay, a sample is defined as positive if the viral genome is detected at threshold cycle (Ct) values ≤35, as However, reproducibility of RNA extraction and RT-PCR might be affected by other factors. We therefore empirically tested the assay sensitivity, when multiple negative samples and one positive sample were mixed at the lysate stage. As shown in Figure 1 , positive samples were readily detected, even when their individual Ct ranged between 35 and 38. Thus, SARS-CoV-2 RNA can be reliably detected in pooled samples without compromising the assay sensitivity.