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

Here An important consideration before implementing group testing is the expected rate of false positive and false negative results. Based on our experience with >2000 samples from asymptomatic individuals, we did not encounter any false positives in the pools, as shown in Figure 1 and Table 1 . False negatives are in principle more worrisome when testing in pools, because samples that failed at the RNA extraction step will be missed (while our individual testing includes amplification of a human transcript serving as an internal control for proper RNA extraction and RT-PCR of each sample). To define the magnitude of this potential problem, we examined a set of 13,781 tests done at our center, which were all expected to show a signal for a human gene serving as internal assay control. Amplification of the human gene failed in 52 samples (0.38% of the cases). Thus, we estimate that our current protocol of pooled sampling carries a risk of missing 0.38% of the positive samples. In a population of 1,000,000 individuals tested, of which 1,000 are positive (rate of 0.1%), this predicts that 4 positive individuals will be missed when using pools. We posit that this is a tolerable situation, particularly given the potentially much higher rates of false negative results due to swab sampling and other errors upstream.