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Updated: Aug 9, 2025
Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots
Published on: February 11, 2022
A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva
Kaitlyn Wagner1,2, Phil Fox1,2, Elizabeth Gordon1,2
1Prion Research Center, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, USA.
Abstract:
In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-h turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.
