Scientific Reports (Feb 2023)

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

  • Kaitlyn Wagner,
  • Phil Fox,
  • Elizabeth Gordon,
  • Westen Hahn,
  • Kenzie Olsen,
  • Alex Markham,
  • Dylan Buglewicz,
  • Platon Selemenakis,
  • Avery Lessard,
  • Daniella Goldstein,
  • Alissa Threatt,
  • Luke Davis,
  • Jake Miller-Dawson,
  • Halie Stockett,
  • Kristin Rugh,
  • Houston Turner,
  • Michelle Remias,
  • Maggie Williams,
  • Jorge Chavez,
  • Gabriel Galindo,
  • Charlotte Cialek,
  • Amanda Koch,
  • Alex Fout,
  • Bailey Fosdick,
  • Bettina Broeckling,
  • Mark D. Zabel

Journal volume & issue
Vol. 13, no. 1
pp. 1 – 17


Read online

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.