LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

PLoS One. 2021 Nov 10;16(11):e0258263. doi: 10.1371/journal.pone.0258263. eCollection 2021.


Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • COVID-19 / virology*
  • DNA Primers / genetics
  • Humans
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Ribonuclease P / genetics*
  • SARS-CoV-2 / genetics*
  • Sensitivity and Specificity
  • Specimen Handling / methods
  • Wastewater / virology*
  • Wastewater-Based Epidemiological Monitoring


  • DNA Primers
  • RNA, Viral
  • Waste Water
  • Ribonuclease P