Enhancing RT-PCR Throughput and Sensitivity through Large-Scale Sample Pooling Using a Nano-Hybrid Membrane.

During the pandemic surge, including SARS-CoV-2 and influenza, pooling samples emerged as an efficient strategy to identify infected individuals in large groups. While pooling enhances RT-PCR throughput, reducing costs and resources, it dilutes positive samples with negative ones, lowering sensitivity and increasing false negatives. This study proposes a new method to address the trade-off between pool sizes and RT-PCR accuracy. This method integrates large-scale pooling with sample enrichment using a nano-hybrid membrane, preventing the pooling-induced decrease in viral concentration and maintaining cycle threshold (Ct) values close to individual positive samples. The nano-hybrid membrane, named SIMPLE (streamlined, simple, and inexpensive method for preconcentration, lysis, and nucleic acid extraction), comprises layered red blood cell membranes, polyethersulfone, and silica membranes. Using SIMPLE, a Ct value reduction to ≈2.6 is demonstrated in pooled COVID-19 samples with a pool size of 6 and found Ct values from larger pool sizes (8, 16, 32, 64, and 128) comparable to individual positive samples.