Developing efficient multiplex qPCR diagnostics using combinatorial pooled testing

The ongoing SARS-CoV-2 pandemic led to significant global increase in molecular diagnostic testing infrastructure and testing kits., including multi-plex PCR kits (e.g. influenza and SARS-CoV-2). However, multiplex-qPCR tests are expensive and therefore not widely used in community settings. To address this challenge, we developed P-BEST – an efficient multiplexed-qPCR solution that is based on combinatorial pooled testing. Unlike traditional pooling methods, P-BEST may be used efficiently for pathogen prevalence >10%. Using a previously developed clinical diagnostic panel for 17 common respiratory and herpes viruses, we optimized a combinatorial pooling design for pooling 282 samples into 94 PCR reactions - a 3-fold reduction in the number of PCR reactions required. Pooling is performed using automated liquid-dispensing robots and positive samples are identified using a decoding algorithm. The pooling method will be used to test a clinical cohort consisting of 1900 samples collected longitudinally from 50 individuals that were swabbed weekly for 9 months. This will allow us to efficiently identify all symptomatic and asymptomatic infections within this cohort. We tested P-BEST using a commercially available multiplex kit (Allplex™-RV7, Seegene). Samples identified using individual testing were pooled, PCR was performed on all pools and P-BEST decoding was applied. P-BEST was able to correctly identify all of the positive samples identified in individual testing. In summary, our method can improve the effectiveness of multiplex diagnostic tests, reduce costs and the number of tests required. We are currently testing the feasibility this approach for community based molecular testing in Clalit clinics in Israel.