Rapid Single‐Round Pool Testing of Infectious Disease Enabled by Multicolor Digital Melting PCR

Abstract Pooled nucleic acid amplification test is a promising strategy to reduce cost and resources for screening large populations for infectious disease. However, the benefit of pooled testing is reversed when disease prevalence is high, because of the need to retest each sample to identify infected individual when a pool is positive. Split, Amplify, and Melt analysis of Pooled Assay (SAMPA) is presented, a multicolor digital melting PCR assay in nanoliter chambers that simultaneously identify infected individuals and quantify their viral loads in a single round of pooled testing. This is achieved by early sample tagging with unique barcodes and pooling, followed by single molecule barcode identification in a digital PCR platform using a highly multiplexed melt curve analysis strategy. The feasibility is demonstrated of SAMPA for quantitative unmixing and variant identification from pools of eight synthetic DNA and RNA samples corresponding to the N1 gene, as well as from heat‐inactivated SARS‐CoV‐2 virus. Single round pooled testing of barcoded samples with SAMPA can be a valuable tool for rapid and scalable population testing of infectious disease.